Reagents and methods for modulating dkk-mediated interactions

ABSTRACT

The present invention provides reagents, compounds, compositions, and methods relating to novel interactions of the extracellular domain of LRP5, HBM (a variant of LRP5), and/or LRP6 with Dkk, including Dkk-1. The various nucleic acids, polypeptides, antibodies, assay methods, diagnostic methods, and methods of treatment of the present invention are related to and impact on Dkk, LRP5, LRP6, HBM, and Wnt signaling. Dkk, LRP5, LRP6, HBM, and Wnt are implicated in bone and lipid cellular signaling. Thus, the present invention provides reagents and methods for modulating lipid levels and/or bone mass and is useful in the treatment and diagnosis of abnormal lipid levels and bone mass disorders, such as osteoporosis.

FIELD OF THE INVENTION

[0001] The present invention relates to signal transduction, bonedevelopment, bone loss disorders, modulation of lipid-relatedconditions, research reagents, methods of screening drug leads, drugdevelopment, treatments for bone and/or lipid disorders, screening anddevelopment of therapies, molecular, cellular, and animal models of boneand/or lipid development and maintenance, which are mediated by Dkk,including Dkk-1, and/or LRP5, LRP6, HBM or other members of the Wntpathway.

BACKGROUND OF THE INVENTION

[0002] Two of the most common types of osteoporosis are postmenopausaland senile osteoporosis. Osteoporosis affects both men and women, and,taken with other abnormalities of bone, presents an ever-increasinghealth risk for an aging population. The most common type ofosteoporosis is that associated with menopause. Most women lose between20-60% of the bone mass in the trabecular compartment of the bone within3-6 years after the cessation of menses. This rapid bone loss isgenerally associated with an increase of bone resorption and formation.However, the resorptive cycle is more dominant and the result is a netloss of bone mass. Osteoporosis is a common and serious disease amongpostmenopausal women. There are an estimated 25 million women in theUnited States alone who are afflicted with this disease. The results ofosteoporosis are personally harmful, and also account for a largeeconomic loss due to its chronicity and the need for extensive andlong-term support (e.g., hospitalization and nursing home care) fromdisease sequelae. This is especially true in elderly patients.Additionally, while osteoporosis is generally not thought of as alife-threatening condition, a 20-30% mortality rate is related to hipfractures in elderly women. A large percentage of this mortality ratecan be directly associated with postmenopausal osteoporosis.

[0003] The most vulnerable tissue in the bone to the effects ofpostmenopausal osteoporosis is the trabecular bone. This tissue is oftenreferred to as spongy bone and is particularly concentrated near theends of the bone, near the joints, and in the vertebrae of the spine.The trabecular tissue is characterized by small structures whichinter-connect with each other as well as the more solid and densecortical tissue which makes up the outer surface and central shaft ofthe bone. This cris-cross network of trabeculae gives lateral support tothe outer cortical structure and is critical to the biomechanicalstrength of the overall structure. In postmenopausal osteoporosis, it isprimarily the net resorption and loss of the trabeculae which lead tothe failure and fracture of the bone. In light of the loss of thetrabeculae in postmenopausal women, it is not surprising that the mostcommon fractures are those associated with bones which are highlydependent on trabecular support, e.g., the vertebrae, the neck of thefemur, and the forearm. Indeed, hip fracture, Colle's fractures, andvertebral crush fractures are indicative of postmenopausal osteoporosis.Osteoporosis affects cortical as well as trabecular bone. Alterations inendosteal bone resorption and Haversian remodeling with age affectcortical thickness and structural integrity contributing the increasedrisk for fracture.

[0004] One of the earliest generally accepted methods for treatment ofpostmenopausal osteoporosis was estrogen replacement therapy. Althoughthis therapy frequently is successful, patient compliance is low,primarily due to the undesirable side-effects of chronic estrogentreatment. Frequently cited side-effects of estrogen replacement therapyinclude reinitiation of menses, bloating, depression, and, potentially,increased risk of breast or uterine cancer. In order to limit the knownthreat of uterine cancer in women who have not had a hysterectomy, aprotocol of estrogen and progestin cyclic therapy is often employed.This protocol is similar to that used in birth control regimens, andoften is not tolerated by women because of the side-effectscharacteristic of progestin. More recently, certain antiestrogens,originally developed for the treatment of breast cancer, have been shownin experimental models of postmenopausal osteoporosis to be efficacious.Among these agents is raloxifene (See, U.S. Pat. No. 5,393,763; Black etal., J. Clin. Invest, 93:63-69 (1994); and Ettinger et al., JAMA282:637-45 (1999)). In addition, tamoxifen, a widely used clinical agentfor treating breast cancer, has been shown to increase bone mineraldensity in post menopausal women suffering from breast cancer (Love etal., N. Engl. J. Med., 326:852-856 (1992)).

[0005] Another therapy for the treatment of postmenopausal osteoporosisis the use of calcitonin. Calcitonin is a naturally occurring peptidewhich inhibits bone resorption and has been approved for this use inmany countries (Overgaard et al., Br. Med. J., 305:556-561 (1992)). Theuse of calcitonin has been somewhat limited, however. Its effects arevery modest in increasing bone mineral density, and the treatment isvery expensive. Another therapy for the treatment of postmenopausalosteoporosis is the use of bisphosphonates. These compounds wereoriginally developed for treating Paget's disease and malignanthypercalcemia. They have been shown to inhibit bone resorption.Alendronate, a bisphosphonate, has been approved for the treatment ofpostmenopausal osteoporosis. These agents may be helpful in thetreatment of osteoporosis, but these agents also have potentialliabilities which include osteomalacia, extremely long half-life in bone(greater than 2 years), and possible “frozen bone syndrome,” e.g., thecessation of normal bone remodeling.

[0006] Senile osteoporosis is similar to postmenopausal osteoporosis inthat it is marked by the loss of bone mineral density and resultingincrease in fracture rate, morbidity, and associated mortality.Generally, it occurs in later life, i.e., after 70 years of age.Historically, senile osteoporosis has been more common in females, butwith the advent of a more elderly male population, this disease isbecoming a major factor in the health of both sexes. It is not clearwhat, if any, role hormones such as testosterone or estrogen have inthis disease, and its etiology remains obscure. Treatment of thisdisease has not been very satisfactory. Hormone therapy, estrogen inwomen and testosterone in men, has shown equivocal results; calcitoninand bisphosphonates may be of some utility.

[0007] The peak mass of the skeleton at maturity is largely undergenetic control. Twin studies have shown that the variance in bone massbetween adult monozygotic twins is smaller than between dizygotic twins(Slemenda et al., J. Bone Miner. Res., 6: 561-567 (1991); Young et al,J. Bone Miner. Res., 6:561-567 (1995); Pocock et al., J. Clin. Invest.,80:706-710 (1987); Kelly et al., J. Bone Miner. Res., 8:11-17 (1993)).It has been estimated that up to 60% or more of the variance in skeletalmass is inherited (Krall et al., J. Bone Miner. Res., 10:S367 (1993)).Peak skeletal mass is the most powerful determinant of bone mass inelderly years (Hui et al., Ann. Int Med., 111:355-361 (1989)), eventhough the rate of age-related bone loss in adult and later life is alsoa strong determinant (Hui et al., Osteoporosis Int., 1:30-34 (1995)).Since bone mass is the principal measurable determinant of fracturerisk, the inherited peak skeletal mass achieved at maturity is animportant determinant of an individual's risk of fracture later in life.Thus, study of the genetic basis of bone mass is of considerableinterest in the etiology of fractures due to osteoporosis.

[0008] Recently, a strong interest in the genetic control of peak bonemass has developed in the field of osteoporosis. The interest hasfocused mainly on candidate genes with suitable polymorphisms to testfor association with variation in bone mass within the normal range, orhas focused on examination of genes and gene loci associated with lowbone mass in the range found in patients with osteoporosis. The vitaminD receptor locus (VDR) (Morrison et al., Nature, 367:284-287 (1994)),PTH gene (Howard et al., J. Clin. Endocrinol. Metab., 80:2800-2805(1995); Johnson et al., J. Bone Miner. Res., 8:11-17 (1995); Gong etal., J. Bone Miner. Res., 10:S462 (1995)) and the estrogen receptor gene(Hosoi et al., J. Bone Miner. Res., 10:S170 (1995); Morrison et al.,Nature, 367:284-287 (1994)) have figured most prominently in this work.These studies are difficult because bone mass (i.e, the phenotype) is acontinuous, quantitative, polygenic trait, and is confounded byenvironmental factors such as nutrition, co-morbid disease, age,physical activity, and other factors. Also, this type of study designrequires large numbers of subjects. In particular, the results of VDRstudies to date have been confusing and contradictory (Garnero et al.,J. Bone Miner. Res., 10:1283-1288 (1995); Eisman et al., J. Bone. Miner.Res., 10:1289-1293 (1995); Peacock, J. Bone Miner. Res., 10: 1294-1297(1995)). Furthermore, thus far, the art has not determined themechanism(s) whereby the genetic influences exert their effect on bonemass.

[0009] While it is well known that peak bone mass is largely determinedby genetic rather than environmental factors, studies to determine thegene loci (and ultimately the genes) linked to variation in bone massare difficult and expensive. Study designs which utilize the power oflinkage analysis, e.g., sib-pair or extended family, are generally moreinformative than simple association studies, although the latter do havevalue. However, genetic linkage studies involving bone mass are hamperedby two major problems. The first problem is the phenotype, as discussedbriefly above. Bone mass is a continuous, quantitative trait, andestablishing a discrete phenotype is difficult. Each anatomical site formeasurement may be influenced by several genes, many of which may bedifferent from site to site. The second problem is the age component ofthe phenotype. By the time an individual can be identified as having lowbone mass, there is a high probability that their parents or othermembers of prior generations will be deceased and therefore unavailablefor study, and younger generations may not have even reached peak bonemass, making their phenotyping uncertain for genetic analysis.

[0010] Thus, there is a need in the art for additional research toolsfor the elucidation of the molecular mechanism of bone modulation, forthe screening and development of candidate drugs, and for treatments ofbone development and bone loss disorders. The present invention isdirected to these, as well as other, important ends.

[0011] In addition to bone modulation, the present invention relates tomodulation of lipid levels. Cardiovascular disease is the most commoncause of mortality in the United States, and atherosclerosis is themajor cause of heart disease and stroke. It is widely appreciated thatcholesterol plays an important role in atherogenesis. Normally, mostcholesterol serves as a structural element in the walls of cells,whereas much of the rest is in transit through the blood or functions asthe starting material for the synthesis of bile acids in the liver,steroid hormones in endocrine cells and vitamin D in skin. The transportof cholesterol and other lipids through the circulatory system isfacilitated by their packaging into lipoprotein carriers. Thesespherical particles comprise protein and phospholipid shells surroundinga core of neutral lipid, including unesterified (“free”) or esterifiedcholesterol and triglycerides. Risk for atherosclerosis increases withincreasing concentrations of low density lipoprotein (LDL) cholesterol,whereas risk is inversely proportional to levels of high-densitylipoprotein (HDL) cholesterol. The receptor-mediated control of plasmaLDL levels has been well-defined, and recent studies have now providednew insights into HDL metabolism.

[0012] The elucidation of LDL metabolism began in 1974 by Michael Brownand Joseph Goldstein. In brief, the liver synthesizes a precursorlipoprotein (very low density lipoprotein, VLDL) that is convertedduring circulation to intermediate density lipoprotein (IDL) and then toLDL. The majority of the LDL receptors expressed in the body are on thesurfaces of liver cells, although virtually all other tissues(“peripheral tissues”) express some LDL receptors. After binding, thereceptor-lipoprotein complex is internalized by the cells via coatedpits and vesicles, and the entire LDL particle is delivered tolysosomes, wherein it is dissembled by enzymatic hydrolysis, releasingcholesterol for subsequent cellular metabolism. This whole-particleuptake pathway is called “receptor-mediated endocytosis.”Cholesterol-mediated feedback regulation of both the levels of LDLreceptors and cellular cholesterol biosynthesis help ensure cellularcholesterol homeostasis. Genetic defects in the LDL receptor in humansresults in familial hypercholesterolemia, a disease characterized byelevated plasma LDL cholesterol and premature atherosclerosis and heartattacks. One hypothesis for the deleterious effects of excess plasma LDLcholesterol is that LDL enters the artery wall, is chemically modified,and then is recognized by a special class of receptors called macrophagescavenger receptors, that mediate the cellular accumulation of the LDLcholesterol in the artery, eventually leading to the formation of anatherosclerotic lesion.

[0013] The major lipoprotein classes include intestinally derivedchylomicrons that transport dietary fats and cholesterol,hepatic-derived VLDL, IDL, and LDL that can be atherogenic, and hepatic-and intestinally-derived HDL that are antiatherogenic. Apoprotein B(ApoB) is necessary for the secretion of chylomicrons (ApoB48) and VLDL,IDL, and LDL (ApoB100). Plasma levels of VLDL triglycerides aredetermined mainly by the rates of secretion in LDL lipolytic activity.Plasma levels of LDL cholesterol are determined mainly by the secretionof ApoB100 into plasma, the efficacy with which VLDL are converted toLDL and by LDL receptor-mediated clearance. Regulation of HDLcholesterol levels is complex and is affected by rates of synthesis ofits Apo proteins, rates of esterification of free cholesterol tocholesterol ester by LCAT, levels of triglyceride-rich lipoproteins andCETP-mediated transfer of cholesterol esters from HDL, and clearancefrom plasma of HDL lipids and Apo proteins.

[0014] Normal lipoprotein transport is associated with low levels oftriglycerides and LDL cholesterol and high levels of HDL cholesterol.When lipoprotein transport is abnormal, lipoprotein levels can change inways that predispose individuals to atherosclerosis and arteriosclerosis(see Ginsburg, Endocrinol. Metab. Clin. North Am., 27:503-19 (1998)).

[0015] Several lipoprotein receptors may be involved in cellular lipiduptake. These receptors include: scavenger receptors; LDLreceptor-related protein/a2-macroglobulin receptor (LRP); LDL receptor;and VLDL receptor. With the exception of the LDL receptor, all of thesereceptors are expressed in atherosclerotic lesions while scavengerreceptors are mostly expressed in macrophages, the LRP and VLDLreceptors may play an important role in mediating lipid uptake in smoothmuscle cells (Hiltunen et al., Atherosclerosis, 137 suppl.:S81-8(1998)).

[0016] A major breakthrough in the pharmacologic treatment ofhypercholesterolemia has been the development of the “statin” class of3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase) inhibitorydrugs. 3-hydroxy-3-methylglutaryl-CoA reductase is the rate controllingenzyme in cholesterol biosynthesis, and its inhibition in the liverstimulates LDL receptor expression. As a consequence, both plasma LDLcholesterol levels and the risk for atherosclerosis decrease. Thediscovery and analysis of the LDL receptor system has had a profoundimpact on cell biology, physiology, and medicine.

[0017] HDL is thought to remove unesterified, or “free” cholesterol (FC)from peripheral tissues, after which most of the cholesterol isconverted to cholesterol ester (CE) by enzymes in the plasma.Subsequently, HDL cholesterol is efficiently delivered directly to theliver and steroidogenic tissues via a selective uptake pathway and theHDL receptor, SR-BI (class B type I scavenger receptor) or, in somespecies, transferred to other lipoproteins for additional transport inmetabolism (see Krieger, Proc. Natl. Acad. Sci. USA, 95:4077-4080(1998)).

[0018] These issues illustrate a need in the art for additional researchtools for the elucidation of the molecular mechanism of lipidmodulation, for the screening and development of candidate drugs, andfor treatments of lipid levels and lipid level modulation disorders. Thepresent invention is directed to these, as well as other, importantends.

SUMMARY OF THE INVENTION

[0019] The present invention provides reagents, compounds, compositionsand methods relating to novel interactions of the extracellular domainof LRP5, HBM (a variant of LRP5), and/or LRP6 with Dkk proteins. LRP5 isalso referred to as Zmax1 or Zmax. Thus, when discussing methods,reagents, compounds, and compositions of the invention which relate tothe interaction between Dkk and LRP5 (or Zmax1), the invention is alsoto be understood to encompass embodiments relating to interactionsbetween Dkk and LRP6 and Dkk and HBM. Moreover, where Dkk is discussedherein, it is to be understood that the methods, reagents, compounds,and compositions of the present invention include the Dkk familymembers, including but not limited to Dkk-1, Dkk-2, Dkk-3, Dkk-4 andSoggy. Furthermore, the invention encompasses novel fragments of Dkk-1which demonstrate a binding interaction between the ligand bindingdomain (LBD) of LRP5 and additional proteins and/or which can modulatean interaction between LRP5, or a variant or fragment thereof, and a Dkkprotein. The invention provides assays, methods, compositions, andcompounds relating to Dkk-Wnt signaling. Numerous Wnt proteins arecompatible with the present invention, including Wnt1-Wnt19, andparticularly, Wnt1, Wnt3, Wnt3a, and Wnt10b. The present inventionfurther provides reagents, compounds, compositions and methodsmodulating interactions between one or more other proteins and Dkk-1.The present invention also provides a series of peptide aptamers whichbind to Dkk-1 or to LRP5 (or HBM and/or LRP6).

[0020] The polypeptides of the invention, for example in the form ofpeptide oligomers, aptamers, proteins, and protein fragments as well asthe nucleic acids of the invention, for example in the form of nucleicacids which encode the polypeptides of the invention as well asantisense, or complimentary nucleic acids, are useful as reagents forthe study of bone mass and lipid level modulation. The polypeptides andnucleic acids of the invention are also useful as therapeutic anddiagnostic agents.

[0021] The present invention provides useful reagents for the modulationof Dkk proteins with LRP5, LRP6, and/or HBM, the modulation Dkk-1 and/orDkk-1 interacting protein activity, and modulation of LRP5/Dkk-1,LRP6/Dkk1 and HBM/Dkk-1 interactions and Dkk-1/Dkk-1 interacting proteininteractions. The present invention provides a series of peptideaptamers which bind Dkk-1 or LRP5, LRP6, and/or HBM.

[0022] An object of the invention is to provide for a method ofregulating LRP5/LRP6/HBM/HBM-like activity in a subject comprisingadministering a therapeutically effective amount of a composition whichmodulates Dkk activity. The subject can be a vertebrate or aninvertebrate organism, but more preferably the organism is a canine, afeline, an ovine, a primate, an equine, a porcine, a caprine, a camelid,an avian, a bovine, or a rodent organism. A more preferred organism is ahuman. In a preferred embodiment, the Dkk protein is Dkk-1. In aparticularly preferred embodiment, Dkk-1 activity is decreased. Inanother embodiment, Dkk activity modulates bone mass and/or lipidlevels. In a preferred embodiment, bone mass is increased and/or lipidlevels are decreased. In another preferred embodiment, the modulation inbone mass is an increase in bone strength determined via one or more ofa decrease in fracture rate, an increase in areal bone density, anincrease in volumetric mineral bone density, an increase in trabecularconnectivity, an increase in trabecular density, an increase in corticaldensity or thickness, an increase in bone diameter, and an increase ininorganic bone content. The invention further provides such a methodwherein the composition comprises a Dkk, Dkk-1 or a LRP5/LRP6/HBMbinding fragment thereof, such as those depicted in FIG. 6 or a mimeticof those fragments depicted in FIG. 6. The invention further providessuch a method wherein the composition comprises one or more of theproteins which interact with Dkk, including Dkk-1, such as thosedepicted in FIG. 5, or a Dkk-binding fragment thereof, or an antisense,siRNA, or shRNA molecule which recognizes and binds to a nucleic acidencoding one or more Dkk interacting or Dkk-1 interacting proteins. Theinvention further provides such a method wherein the compositioncomprises an LRP5/LRP6/Zmax1 antibody, Dkk antibody, a Dkk-1 antibody oran antibody to a Dkk-1 interacting protein. The invention furtherprovides such a method wherein the compositions comprise an aptamer ofDkk or Dkk-1, such as those depicted in FIG. 3 (SEQ ID NOs:171-188), ora mimetic of such an aptamer. The method further provides that inventionfurther provides such a method wherein the compositions comprise anaptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimeticof such an aptamer.

[0023] A composition of the present invention may modulate activityeither by enhancing or inhibiting the binding of Dkk to LRP5/LRP6/Zmax1,particularly Dkk-1, or the binding of Dkk-1 to a Dkk-1 interactingprotein, such as those shown in FIG. 5. A composition of the presentinvention may comprise an LRP5 peptide aptamer, such as OST262 (SEQ IDNO:208), FIGS. 4 (SEQ ID NOs:189-192) (particularly, peptide (SEQ IDNO:191) and 13 (including SEQ ID NOs:204-214), or a mimetic of such anaptamer. Preferred compositions of the present invention also compriseLRP5 antibodies.

[0024] Another aspect of the invention is to provide for a method ofregulating Dkk-Wnt pathway activity in a subject comprisingadministering a therapeutically effective amount of a composition whichmodulates Dkk-Wnt pathway activity. In a preferred embodiment, the Dkkprotein is Dkk-1. In a particularly preferred embodiment, Dkk-1 activityis decreased. In another embodiment, Dkk activity modulates bone massand/or lipid levels. In a preferred embodiment, bone mass is increasedand/or lipid levels are decreased. In another preferred embodiment, themodulation in bone mass is an increase in bone strength determined viaone or more of a decrease in fracture rate, an increase in areal bonedensity, an increase in volumetric mineral bone density, an increase intrabecular connectivity, an increase in trabecular density, an increasein cortical density or thickness, an increase in bone diameter, and anincrease in inorganic bone content. In another preferred embodiment, theWnt is Wnt1-Wnt19. In a particularly preferred embodiment, the Wnt isWnt1, Wn3, Wnt3a, or Wnt10b. Preferred compositions compriseDkk-modulating or Dkk-1-modulating compounds or one or more Dkkinteracting or Dkk-1 interacting proteins, or a Dkk-binding fragmentthereof. Other preferred Dkk modulating compositions comprise a Dkk orDkk-1 antibody or an antibody to a Dkk interacting or Dkk-1 interactingprotein. Also contemplated are antisense, siRNA, and shRNA moleculeswhich recognize and bind to a nucleic acid encoding one or more Dkk-1interacting proteins. The invention further provides such a methodwherein the composition comprises a biologically active or LRP5/LRP6/HBMbinding fragment of Dkk, including Dkk-1, such as those depicted in FIG.6 or a mimetic of those fragments depicted in FIG. 6. The Dkk modulatingcomposition may also comprise a peptide aptamer of a Dkk interacting orDkk-1 interacting protein, or a mimetic of such an aptamer. Acomposition of the present invention may modulate activity either byenhancing or inhibiting the binding of Dkk, including Dkk-1, to LRP5,LRP6, or HBM or the binding of Dkk, including Dkk-1, to a Dkkinteracting protein, such as those shown in FIG. 5. The inventionfurther provides such a method wherein the composition comprises anaptamer of Dkk or Dkk-1, such as those depicted. A composition of thepresent invention may comprise an LRP5 peptide aptamer, such as OST262(SEQ ID NO:208). Preferred compositions of the present invention alsocomprise LRP5 antibodies.

[0025] A further aspect of the invention is to provide for a method ofmodulating Wnt signaling in a subject comprising administering atherapeutically effective amount of a composition which modulates Dkkactivity or modulates Dkk interaction with LRP5 (or LRP6 or HBM). In apreferred embodiment, the Dkk protein is Dkk-1. In a particularlypreferred embodiment, Dkk-1 activity is decreased. In anotherembodiment, Dkk activity modulates bone mass and/or lipid levels. In apreferred embodiment, bone mass is increased and/or lipid levels aredecreased. In another preferred embodiment, the modulation in bone massis an increase in bone strength determined via one or more of a decreasein fracture rate, an increase in areal bone density, an increase involumetric mineral bone density, an increase in trabecular connectivity,an increase in trabecular density, an increase in cortical density orthickness, an increase in bone diameter, and an increase in inorganicbone content. In another preferred embodiment, the Wnt is Wnt1-Wnt19. Ina particularly preferred embodiment, the Wnt is Wnt1, Wnt3, Wnt3a, orWnt10b. Preferred Wnt modulating compositions comprise one or more Dkkinteracting or Dkk-1 interacting proteins, or a biologically active orLRP5/LRP6/HBM binding fragment thereof. Also contemplated are antisense,siRNA, and shRNA molecules which recognize and bind to a nucleic acidencoding one or more Dkk interacting or Dkk-1 interacting proteins. Theinvention further provides such a method wherein the compositioncomprises a biologically active or LRP5/LRP6/HBM binding fragment of Dkkor Dkk-1, such as those depicted in FIG. 6 or a mimetic of thosefragments depicted in FIG. 6. The Dkk modulating composition may alsocomprise a peptide aptamer of a Dkk interacting or Dkk-1 interactingprotein, or a mimetic of such an aptamer. A composition of the presentinvention may modulate activity either by enhancing or blocking thebinding of Dkk, including Dkk-1, to LRP5, LRP6, or HBM or the binding ofDkk or Dkk-1 to a Dkk interacting or Dkk-1 interacting protein, such asthose shown in FIG. 5. The invention further provides such a methodwherein compositions comprising an aptamer of Dkk or Dkk-1, such asthose depicted in FIG. 3 (SEQ ID NOs:171-188), or a mimetic of such anaptamer. The invention further provides such a method wherein thecomposition comprises a Dkk or Dkk-1 antibody or an antibody to a Dkkinteracting or Dkk-1 interacting protein. The invention further providessuch a method wherein compositions of an LRP5 peptide aptamer, such asOST262 (SEQ ID NO:208), FIG. 4 (SEQ ID NO:189-192 (particularly peptide(SEQ ID NO:191) and FIG. 13 (including SEQ ID NOs:204-214), or a mimeticof such an aptamer. Additional preferred compositions of the presentinvention also comprise LRP5 antibodies.

[0026] Additionally, the invention provides for a method of modulatingbone mass and/or lipid levels in a subject comprising administering tothe subject a composition which modulates Dkk activity or Dkkinteraction with LRP5 in an amount effective to modulate bone massand/or lipid levels, wherein bone mass and/or lipid levels are in needof modulation. In a preferred embodiment, the Dkk protein is Dkk-1. In aparticularly preferred embodiment, Dkk-1 activity is decreased. Inanother embodiment, Dkk activity modulates bone mass and/or lipidlevels. In a preferred embodiment, bone mass is increased and/or lipidlevels are decreased. In another preferred embodiment, the modulation inbone mass is an increase in bone strength determined via one or more ofa decrease in fracture rate, an increase in areal bone density, anincrease in volumetric mineral bone density, an increase in trabecularconnectivity, an increase in trabecular density, an increase in corticaldensity or thickness, an increase in bone diameter, and an increase ininorganic bone content. Preferred bone mass and/or lipid modulatingcompositions comprise one or more Dkk interacting or Dkk-1 interactingproteins, or a biologically active or LRP5/LRP6/HBM binding fragmentthereof. Also contemplated are antisense, siRNA, and shRNA moleculeswhich recognize and bind to a nucleic acid encoding one or more Dkkinteracting or Dkk-1 interacting proteins. The invention furtherprovides such a method wherein the composition comprises a biologicallyactive or LRP5/LRP6/HBM binding fragment of Dkk, including Dkk-1, suchas those depicted in FIG. 6 or a mimetic of those fragments depicted inFIG. 6. The Dkk modulating composition may also comprise a peptideaptamer of a Dkk interacting or Dkk-1 interacting protein, or a mimeticof such an aptamer. The invention further provides such a method whereinthe composition comprises an aptamer of Dkk or Dkk-1, such as thosedepicted in FIG. 3 (SEQ ID NOs:171-188), or a mimetic of such anaptamer. A composition of the present invention may modulate activityeither by enhancing or inhibiting the binding of Dkk, including Dkk-1,to LRP5, LRP6, or HBM or the binding of Dkk, including Dkk-1, to a Dkkinteracting protein, such as those shown in FIG. 5. The inventionfurther provides such a method wherein the composition comprises a Dkkor Dkk-1 antibody or an antibody to a Dkk interacting or Dkk-1interacting protein. A composition of the present invention may comprisean LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), FIGS. 4 (SEQ IDNOs:189-192 (particularly peptide 13 (SEQ ID NO:191)) and 13 (includingSEQ ID NOs:204-214), or a mimetic of such an aptamer. Preferredcompositions of the present invention also comprise LRP5 antibodies. Itis a further aspect of the invention that such lipid-modulated diseasesinclude a cardiac condition, atherosclerosis, familial lipoproteinlipase deficiency, familial apoprotein CII deficiency, familial type 3hyperlipoproteinemia, familial hypercholesterolemia, familialhypertriglyceridemia, multiple lipoprotein-type hyperlipidemia, elevatedlipid levels due to dialysis and/or diabetes, and an elevated lipidlevel of unknown etiology.

[0027] Bone disorders contemplated for treatment and/or diagnosis by themethods and compositions disclosed herein include a bone developmentdisorder, a bone fracture, age related loss of bone, a chondrodystrophy,a drug-induced bone disorder, high bone turnover, hypercalcemia,hyperostosis, osteogenesis imperfecta, osteomalacia, osteomyelitis,osteoporosis, Paget's disease, osteoarthritis, and rickets.

[0028] It is a further object of the invention to provide a method ofscreening for compounds or compositions which modulates the interactionof Dkk with LRP5, LRP6, HBM, or a Dkk-binding fragment of LRP5, LRP6, orHBM comprising:

[0029] (a) exposing Dkk or a LRP5/LRP6/HBM binding fragment thereof to acompound; and

[0030] (b) determining whether said compound binds to Dkk or theLRP5/LRP6/HBM binding fragment thereof.

[0031] In a preferred embodiment, the Dkk is Dkk-1. In a particularlypreferred embodiment, the binding of Dkk-1 to LRP5/LRP6/HBM isdecreased.

[0032] It is a further object of the invention to provide a method ofscreening compounds or compositions which modulate the interaction ofDKK with LRP5, LRP6, HBM, or a DKK-finding fragment thereof comprising:

[0033] (a) exposing DKK or a LRP5/LRP6/HBM binding fragment thereof to acompound; and,

[0034] (b) determining whether said compound modulates the interactionof Dkk with LRP5, LRP6, or HBM, or the Dkk-binding fragment ofLRP5/LRP6/HBM.

[0035] In a preferred embodiment, the Dkk is Dkk-1. In a particularlypreferred embodiment, the interaction of Dkk-1 with LRP5/LRP6/HBM isdecreased.

[0036] It is a further object of the invention to provide a method ofscreening for compounds or compositions which modulates the interactionof Dkk with LRP5, LRP6, HBM, or a Dkk-binding fragment of LRP5, LRP6, orHBM comprising:

[0037] (a) exposing Dkk or a LRP5/LRP6/HBM binding fragment thereof to acompound;

[0038] (b) determining whether said compound binds to Dkk or theLRP5/LRP6/HBM binding fragment thereof; and,

[0039] (c) further determining whether said compound modulates theinteraction of Dkk with LRP5, LRP6, or HBM, or the Dkk-binding fragmentof LRP5/LRP6/HBM.

[0040] In preferred embodiments of such methods, Dkk or a biologicallyactive fragment thereof is attached to a solid substrate. In analternative embodiment of the invention, LRP5/LRP6/HBM, or abiologically active fragment thereof (such as the ligand bindingdomain), is exposed to the compound. Another aspect of the inventionprovides for compounds and compositions identified by the disclosedmethods. A preferred embodiment of the invention provides that thecompound screened in an afore-mentioned method is one or more proteinswhich interact with Dkk, particularly Dkk-1, as depicted in FIG. 5, or aLRP5/LRP6/HBM-binding fragment thereof. Another preferred embodimentprovides that the compound comprises a Dkk or Dkk-1 peptide aptamer,such as those depicted in FIG. 3 (SEQ ID NOs:171-188), or a mimetic ofsuch aptamers. The compound may also comprise a peptide aptamer of a Dkkinteracting or Dkk-1 interacting protein, or a mimetic of such anaptamer. The method further provides that the compound comprises a Dkkor Dkk-1 antibody or an antibody to a Dkk-1 interacting protein. Theinvention further provides that the compound may comprise an LRP5peptide aptamer, such as OST262 (SEQ ID NO:208), FIG. 4 (SEQ IDNOs:189-192) (particularly peptide 13 (SEQ ID NO:191)) and FIG. 13(including SEQ ID NOs:204-214), or a mimetic of such an aptamer.Preferred compounds of the present invention also comprise LRP5antibodies.

[0041] It is a further object of the invention to provide a method ofscreening for compounds or compositions which modulate the interactionof Dkk and a Dkk interacting protein comprising:

[0042] (a) exposing a Dkk interacting proteins or a Dkk-binding fragmentthereof to a compound; and,

[0043] (b) determining whether said compound binds to a Dkk interactingproteins or the Dkk-binding fragment thereof.

[0044] In a preferred embodiment, the Dkk is Dkk-1.

[0045] It is a further object of the invention to provide a method ofscreening for compounds or compositions which modulate the interactionof Dkk and a Dkk interacting protein comprising:

[0046] (a) exposing Dkk interacting protein(s) or a Dkk-binding fragmentthereof to a compounds; and,

[0047] (b) determining whether said compound modulates the interactionof Dkk and Dkk interacting proteins.

[0048] It is a further object of the invention to provide a method ofscreening for compounds or compositions which modulate the interactionof Dkk and a Dkk interacting protein comprising:

[0049] (a) exposing a Dkk interacting proteins or a Dkk-binding fragmentthereof to a compound;

[0050] (b) determining whether said compound binds to a Dkk interactingproteins or the Dkk-binding fragment thereof; and,

[0051] (c) further determining whether said compound modulates theinteraction of Dkk and Dkk interacting proteins.

[0052] In a preferred embodiment, Dkk is Dkk-1.

[0053] In preferred embodiments of such methods, the Dkk interactingproteins, particularly Dkk-1 interacting proteins, or a Dkk-bindingfragment thereof are attached to a solid substrate. Another aspect ofthe invention provides for compounds and compositions identified by thedisclosed methods. A preferred embodiment provides that the compoundcomprises a Dkk or Dkk-1 peptide aptamer, such as those depicted in FIG.3 (SEQ ID NOs:171-188), or a mimetic of such aptamers. The compound mayalso comprise a peptide aptamer of a Dkk interacting or Dkk-1interacting protein, or a mimetic of such an aptamer. The compound mayalso comprise an antibody to a Dkk interacting or Dkk-1 interactingprotein.

[0054] It is another object of the invention to provide for acomposition for treating bone mass disorders comprising a LRP5/LRP6/HBMmodulating compound and a pharmaceutically acceptable excipient and/orcarrier therefor. Preferred LRP5 (or LRP6 or HBM) modulating compoundsinclude Dkk or Dkk-1 or a LRP5/LRP6/HBM binding fragment thereof. Alsocontemplated are compounds which comprise monoclonal or polyclonalantibodies or immunologically active fragments thereof which bind toDkk, including Dkk-1, and a pharmaceutically acceptable excipient and/orcarrier. Another preferred embodiment provides that the modulatingcompound comprises one or more Dkk interacting or Dkk-1 interactingproteins, or a biologically active fragment thereof. Also contemplatedare compounds which comprise monoclonal or polyclonal antibodies orimmunologically active fragments thereof which bind to Dkk interactingor Dkk-1 interacting proteins, or a biologically active fragmentthereof, and a pharmaceutically acceptable excipient and/or carrier.Another preferred embodiment provides that the modulating compoundcomprises an antisense, siRNA, and shRNA molecule which recognizes andbinds to a nucleic acid encoding a Dkk interacting or Dkk-1 interactingprotein. Another preferred embodiment provides that the modulatingcompound comprises a Dkk or Dkk-1 peptide aptamer, a mimetic of a Dkk orDkk-1 peptide aptamer, a peptide aptamer of a Dkk interacting or Dkk-1interacting protein, or a mimetic of such an aptamer. Another embodimentprovides that the compound comprises an LRP5 peptide aptamer, such asOST262 (SEQ ID NO:208), FIG. 4 (SEQ ID NOs:189-192) (particularlypeptide) and FIG. 13 (including SEQ ID NOs:204-214), or a mimetic ofsuch an aptamer. Preferred compounds of the present invention alsocomprise LRP5 antibodies.

[0055] It is a further object of the invention to provide apharmaceutical composition for treating a Dkk-mediated disease orcondition comprising a compound which modulates Dkk activity and acarrier therefor, including pharmaceutically acceptable excipients. Suchcompositions include those wherein the compound comprises an antisense,siRNA, and shRNA molecule or an antibody which binds to Dkk, includingDkk-1, and thereby prevents it from interacting with LRP5, LRP6, or HBM.Other such compositions include one or more of Dkk interacting or Dkk-1interacting proteins, such as those depicted in FIG. 5, or a Dkk-bindingfragment thereof, or a monoclonal or polyclonal antibody, orimmunologically active fragment thereof, which binds to a Dkkinteracting or Dkk-1 interacting protein or Dkk-binding fragmentthereof. Other contemplated compositions include antisense, siRNA, andshRNA molecules which recognize and bind to a nucleic acid encoding aDkk interacting or Dkk-1 interacting protein. Further contemplatedcompositions include Dkk and Dkk-1 peptide aptamers, such as thosedepicted in FIG. 3 (SEQ ID NOs;171-188), mimetics of such aptamers, apeptide aptamer of a Dkk interacting or Dkk-1 interacting protein, or amimetic of such an aptamer. Other contemplated compositions comprise anLRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), FIG. 4 (SEQ IDNOs:189-192) (particularly peptide 13 (SEQ ID NO:191)) and FIG. 13(including SEQ ID NO:204-214), or a mimetic of such an aptamer. Otherpreferred compositions of the present invention comprise LRP5antibodies.

[0056] A further object of the invention to provide for a method ofmodulating the expression of a nucleic acid encoding a Dkk interactingor Dkk-1 interacting protein in an organism, such as those shown in FIG.5, comprising the step of administering to the organism an effectiveamount of composition which modulates the expression of a nucleic acidencoding a Dkk-1 interacting protein. In a preferred embodiment, saidcomposition comprises an antisense, siRNA, or shRNA molecule whichrecognizes and binds to a nucleic acid encoding a Dkk interacting orDkk-1 interacting protein.

[0057] One aspect of the invention provides for a method of modulatingat least one activity of Dkk or a Dkk-1 interacting protein comprisingadministering an effective amount of a composition which modulates atleast one activity of Dkk or a Dkk-1 interacting protein. The inventionprovides for a composition comprising a Dkk interacting or Dkk-1interacting protein, such as those shown in FIG. 5, or a biologicallyactive fragment thereof. Other agents contemplated for this method areantisense, siRNA, or shRNA molecules which recognize and bind to anucleic acid encoding a Dkk interacting or Dkk-1 interacting protein.The method further provides that the composition comprises a Dkk orDkk-1 antibody or an antibody to a Dkk interacing or Dkk-1 interactingprotein. In another preferred embodiment, the composition comprises aDkk or Dkk-1 peptide aptamer, a mimetic of a Dkk or Dkk-1 peptideaptamer, a peptide aptamer of a Dkk interacting or Dkk-1 interactingprotein, or a mimetic of such an aptamer. The method provides that acomposition of the present invention may comprise an LRP5 peptideaptamer, such as OST262 (SEQ ID NO:208), FIG. 4 (SEQ ID NO:189-192)(particularly peptide including (SEQ ID NO:191)) and Figure including(SEQ ID NOs:204-214), or a mimetic of such an aptamer. Preferredcompositions of the present invention also comprise LRP5 antibodies. Ina further preferred embodiment, the modulated Dkk activity is lipidmodulation or bone mass modulation.

[0058] In all of the testing/screening embodiments of the presentinvention discussed below to obtain compounds or compositions whichultimately impact LRP5/LRP6/HBM signaling, one skilled in the art willrecognize that HBM can be used as a control in the absence of a testsample or compound. Further, the effect of a test sample of compound onWnt signaling through the interaction of Dkk with LRP5/LRP6/HBM does notnecessarily require a direct measurement of an association orinteraction of Dkk and LRP5/LRP6/HBM. Other positivephenotypes/activities established by the High Bone Mass phenotype or byusing HBM as a control.

[0059] One aspect of the invention provides for a method of identifyingbinding partners for a Dkk protein comprising the steps of:

[0060] (a) exposing the Dkk protein(s) or a LRP5/LRP6 binding fragmentthereof to a potential binding partner; and

[0061] (b) determining if the potential binding partner binds to a Dkkprotein or the LRP5/LRP6 binding fragment thereof.

[0062] In a preferred embodiment, the Dkk is Dkk-1.

[0063] Another aspect of the invention is to provide for a method ofidentifying a compound that effects Dkk-mediated activity comprising

[0064] (a) providing a group of transgenic animals having (1) aregulatable one or more Dkk interacting protein genes, (2) a knock-outof one or more Dkk interacting protein genes, or (3) a knock-in of oneor more Dkk interacting protein genes;

[0065] (b) providing a second group of control animals respectively forthe group of transgenic animals in step (a); and

[0066] (c) exposing the transgenic animal group and the control animalgroup to a potential Dkk-modulating compound which modulates bone massor lipid levels; and

[0067] (d) comparing the transgenic animal group and the control animalgroup and determining the effect of the compound on bone mass or lipidlevels in the transgenic animals as compared to the control animals.

[0068] In a preferred embodiment, the Dkk is Dkk-1.

[0069] It is another aspect of the invention to provide for a method fordetermining whether a compound modulates a Dkk interacting protein, saidmethod comprising the steps of:

[0070] (a) mixing the Dkk interacting protein or a Dkk-binding fragmentthereof with the ligand binding domain of Dkk in the presence of said atleast one compound;

[0071] (b) measuring the amount of said binding domain of Dkk bound tosaid Dkk interacting protein or the Dkk-binding fragment thereof ascompared to a control without said at least one compound; and

[0072] (c) determining whether the compound reduces the amount of saidbinding domain of Dkk binding to said Dkk interacting protein orDkk-binding fragment thereof.

[0073] In a preferred embodiment, the Dkk is Dkk-1.

[0074] In a preferred embodiment, the binding domain is attached to asolid substrate. The invention further provides for compounds identifiedby this method. In a preferred embodiment, the invention provides thatthe Dkk interacting or Dkk-1 interacting protein is detected byantibodies. In another preferred embodiment, the solid substrate is amicroarray. Another preferred embodiment provides that the ligandbinding domain of Dkk and/or Dkk interacting protein is fused orconjugated to a peptide or protein. The invention also provides that thecompounds include Dkk and Dkk-1 peptide aptamers, mimetics of Dkk andDkk-1 peptide aptamers, Dkk and Dkk-1 interacting proteins peptideaptamers, or mimetics of such aptamers.

[0075] An aspect of the invention provides a composition comprising oneor more polypeptide sequences of one or more Dkk-1 interacting proteins,or a biologically active fragment thereof, one or more Dkk proteins, ora biologically active fragment thereof, or LRP5/LRP6/HBM polypeptidesequences or a biologically active fragment thereof (for example, theligand binding domain) and a pharmaceutically acceptable excipientand/or carrier. Another aspect of the invention provides that thecomposition comprises a Dkk or Dkk-1 antibody or an antibody to a Dkkinteracting or Dkk-1 interacting protein and a pharmaceuticallyacceptable excipient. A composition of the present invention maycomprise an LRP5 peptide aptamer, such as OST262 (SEQ ID NO:208), FIG. 4(SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191)) and FIG.13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer. Acomposition of the present invention may comprise a Dkk peptide aptamer,for example as shown in FIG. 3 (SEQ ID NOs:171-188). Preferredcompositions of the present invention also comprise LRP5 antibodies.

[0076] Another aspect of the invention is to provide an antibody orimmunologically active antibody fragment which recognizes and binds to aDkk-1 amino acid sequence selected from the group consisting of:Asn34-His266 (SEQ ID NO:1 10), Asn34-Cys245 (SEQ ID NO:111),Asn34-Lys182 (SEQ ID NO:112), Cys97-His266 (SEQ ID NO:113),Val139-His266 (SEQ ID NO:114), Gly183-His266 (SEQ ID NO:115),Cys97-Cys245 (SEQ ID NO:116), or Val139-Cys245 (SEQ ID NO:117) of humanDkk-1. Additional antibodies may bind to any of the sequences depictedin FIG. 3 (SEQ ID NOs:171-188) and FIG. 4 (SEQ ID NOs:189-192). Anotheraspect of the invention is to provide for polyclonal antibodies to oneor more amino acid sequences: Peptide 1—GNKYQTIDNYQPYPC (SEQ ID NO:118),Peptide 2—LDGYSRRTTLSSKMYHTKGQEG (SEQ ID NO:119), Peptide3—RIQKDHHQASNSSRLHTCQRH (SEQ ID NO:120), Peptide 4—RGEIEETITESFGND (SEQID NO:121), and Peptide 5—EIFQRCYCGEGLSCRIQKD (SEQ ID NO: 122).

[0077] It is a further object of the invention to provide a nucleic acidencoding a Dkk protein, e.g. Dkk-1, a Dkk interacting or Dkk-1interacting protein aptamer, or an LRP5 aptamer comprising a nucleicacid encoding a scaffold protein in-frame with the activation domain ofGal4 or LexA that is in-frame with a nucleic acid which encodes for aDkk or Dkk-1 or Dkk interacting or Dkk-1 interacting protein amino acidsequence. Preferably the scaffold protein is thioredoxin (trxA), S1nuclease from Staphylococcus or M13. Other preferable embodimentsinclude Dkk-1 amino acid sequences selected from FIG. 6.

[0078] It is yet a further object of the invention to provide acomposition comprising a polypeptide sequence of FIG. 3 (SEQ IDNOs:171-188), FIG. 4 (SEQ ID NO:189-192), or of Dkk-1 interactingproteins identified in FIG. 5 and a pharmaceutically acceptableexcipient and/or carrier.

[0079] Another aspect of the invention includes a method of detectingthe modulatory activity of a compound on the binding interaction of afirst peptide and a second peptide of a peptide binding pair that bindthrough extracellular interaction in their natural environment,comprising:

[0080] (i) culturing at least one eukaryotic cell, wherein theeukaryotic cell comprises;

[0081] a) a nucleotide sequence encoding a first heterologous fusionprotein comprising the first peptide or a segment thereof joined to aDNA binding domain of a transcriptional activation protein;

[0082] b) a nucleotide sequence encoding a second heterologous fusionprotein comprising the second peptide or a segment thereof joined to atranscriptional activation domain of a transcriptional activationprotein;

[0083] wherein binding of the first peptide or segment thereof and thesecond peptide or segment thereof reconstitutes a transcriptionalactivation protein; and

[0084] c) a reporter element activated under positive transcriptionalcontrol of the reconstituted transcriptional activation protein, whereinexpression of the reporter element produces a selected phenotype;

[0085] (ii) incubating a compound with the eukaryotic cell underconditions suitable to detect the selected phenotype; and

[0086] (iii) detecting the ability of the compound to affect the bindinginteraction of the peptide binding pair by determining whether thecompound affects the expression of the reporter element which producesthe selected phenotype;

[0087] wherein (1) said first peptide is a Dkk peptide and said secondpeptide is a peptide selected from LRP5, HBM, LRP6, and the Dkk-bindingportion of LRP5/LRP6/HBM or (2) said first peptide is a Dkk-interactingprotein or the Dkk-binding fragment thereof, and said second peptide isa Dkk peptide.

[0088] In one embodiment, the eukaryotic cell is a yeast cell. In apreferred embodiment, the yeast cell is Saccharomyces. In a particularlypreferred embodiment, the Saccharomyces cell is Saccharomycescerevisiae. The invention further provides that the compound maycomprise a Dkk interacting or Dkk-1 interacting protein, or abiologically active fragment thereof. In one embodiment, the Dkkinteracting or Dkk-1 interacting protein, or a Dkk-binding fragmentthereof, is added directly to the assay. In another embodiment, the Dkkinteracting or Dkk-1 interacting protein, or a Dkk-binding fragmentthereof, is recombinantly expressed by the eukaryotic cell in additionto the first and second peptides. In a preferred embodiment the compoundcomprises a Dkk or Dkk-1 aptamer, a mimetic of a Dkk or Dkk-1 peptideaptamer, a Dkk interacting or Dkk-1 interacting protein aptamer, or amimetic of a Dkk-1 interacting protein aptamer. Other preferredembodiments provide that the compound comprises an LRP5 peptide aptamer,such as OST262 (SEQ ID NO:208), FIG. 4 (SEQ ID NOs:189-192)(particularly peptide 13 (SEQ ID NO:191) and FIG. 13 (including SEQ IDNOs:204-214), or a mimetic of such an aptamer. Alternatively, thepresent invention also provides that the compound may comprise LRP5antibodies or Dkk antibodies. In another embodiment, the yeast cellfurther comprises at least one endogenous nucleotide sequence selectedfrom the group consisting of a nucleotide sequence encoding the DNAbinding domain of a transcriptional activation protein, a nucleotidesequence encoding the transcriptional activation domain of atranscriptional activation protein, and a nucleotide sequence encodingthe reporter element, wherein at least one of the endogenous nucleotidesequences is inactivated by mutation or deletion. In another embodiment,the peptide binding pair comprises a ligand and a receptor to which theligand binds. In one embodiment, the transcriptional activation proteinis Gal4, Gcn4, Hap1, Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81,Lac9, Qa1F, VP16, or a mammalian nuclear receptor. In anotherembodiment, at least one of the heterologous fusion proteins isexpressed from an autonomously-replicating plasmid. In one embodiment,the DNA binding domain comprises a heterologous DNA-binding domain of atranscriptional activation protein. In a preferred embodiment, the DNAbinding protein is selected from the group consisting of a mammaliansteroid receptor and bacterial LexA protein. In another embodiment, thereporter element is selected from the group consisting of lacZ, apolynucleotide encoding luciferase, a polynucleotide encoding greenfluorescent protein (GFP), and a polynucleotide encoding chloramphenicolacetyltransferase. In a particularly preferred embodiment, the reporterelement is lacZ

[0089] The invention further provides for a rescue screen for detectingthe activity of a compound for modulating the binding interaction of afirst peptide and a second peptide of a peptide binding pair,comprising:

[0090] (i) culturing at least one yeast cell, wherein the yeast cellcomprises;

[0091] a) a nucleotide sequence encoding a first heterologous fusionprotein comprising the first peptide or a segment thereof joined to aDNA binding domain of a transcriptional activation protein;

[0092] b) a nucleotide sequence encoding a second heterologous fusionprotein comprising the second peptide or a segment thereof joined to atranscriptional activation domain of a transcriptional activationprotein;

[0093] wherein binding of the first peptide or segment thereof and thesecond peptide or segment thereof reconstitutes a transcriptionalactivation protein; and

[0094] c) a reporter element activated under positive transcriptionalcontrol of the reconstituted transcriptional activation protein, whereinexpression of the reporter gene prevents exhibition of a selectedphenotype;

[0095] (ii) incubating a compound with the yeast cell under conditionssuitable to detect the selected phenotype; and

[0096] (iii) detecting the ability of the compound to affect the bindinginteraction of the peptide binding pair by determining whether thecompound affects the expression of the reporter element which preventsexhibition of the selected phenotype,

[0097] wherein said first peptide is a Dkk peptide and said secondpeptide is a peptide selected from LRP5, HBM, LRP6 and a Dkk-bindingfragment of LRP5/LRP6/HBM.

[0098] In a preferred embodiment, the invention provides that the yeastcell is Saccharomyces. In a particularly preferred embodiment, theSaccharomyces cell is Saccharomyces cerevisiae. In one embodiment, thecompound comprises one or more Dkk interacting or Dkk-1 interactingproteins, or a Dkk-binding fragment thereof. Compounds used in thepresent invention may comprise an LRP5 peptide aptamer, such as OST262(SEQ ID NO:208), FIG. 4 (SEQ ID NOs:189-192) (particularly peptide 13(SEQ ID NO:191)) and FIG. 13 (including SEQ ID NOs:204-214), or amimetic of such an aptamer. Alternatively, the compound may compriseLRP5 antibodies or Dkk antibodies. In another embodiment, the yeast cellfurther comprises at least one endogenous nucleotide sequence selectedfrom the group consisting of a nucleotide sequence encoding the DNAbinding domain of a transcriptional activation protein, a nucleotidesequence encoding the transcriptional activation domain of atranscriptional activation protein, and a nucleotide sequence encodingthe reporter gene, wherein at least one of the endogenous nucleotidesequences is inactivated by mutation or deletion. In another embodiment,the transcriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5,Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammaliannuclear receptor. In one embodiment, at least one of the heterologousfusion proteins is expressed from an autonomously-replicating plasmid.In another embodiment, the DNA binding domain is a heterologousDNA-binding domain of a transcriptional activation protein.

[0099] The invention also provides for a rescue screen for detecting themodulatory activity of a compound on the binding interaction of a firstpeptide and a second peptide of a peptide binding pair, comprising:

[0100] (i) culturing at least one yeast cell, wherein the yeast cellcomprises;

[0101] a) a nucleotide sequence encoding a first heterologous fusionprotein comprising the first peptide or a segment thereof joined to aDNA binding domain of a transcriptional activation protein;

[0102] b) a nucleotide sequence encoding a second heterologous fusionprotein comprising the second peptide or a segment thereof joined to atranscriptional activation domain of a transcriptional activationprotein;

[0103] wherein binding of the first peptide or segment thereof and thesecond peptide or segment thereof reconstitutes a transcriptionalactivation protein; and

[0104] c) a reporter element activated under positive transcriptionalcontrol of the reconstituted transcriptional activation protein, whereinexpression of the reporter element prevents exhibition of a selectedphenotype;

[0105] (ii) incubating a compound with the yeast cell under conditionssuitable to detect the selected phenotype; and

[0106] (iii) detecting the ability of the compound to affect the bindinginteraction of the peptide binding pair by determining whether thecompound affects the expression of the reporter element which preventsexhibition of the selected phenotype,

[0107] wherein said first peptide is a Dkk interacting or Dkk-1interacting protein peptide and said second peptide is a Dkk or Dkk-1peptide.

[0108] In a preferred embodiment of the rescue screen, the yeast cell isSaccharomyces. In a particularly preferred embodiment, the Saccharomycescell is Saccharomyces cerevisiae. In another embodiment, the yeast cellfurther comprises at least one endogenous nucleotide sequence selectedfrom the group consisting of a nucleotide sequence encoding the DNAbinding domain of a transcriptional activation protein, a nucleotidesequence encoding the transcriptional activation domain of atranscriptional activation protein, and a nucleotide sequence encodingthe reporter gene, wherein at least one of the endogenous nucleotidesequences is inactivated by mutation or deletion. In one embodiment, thetranscriptional activation protein is Gal4, Gcn4, Hap1, Adr1, Swi5,Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, Qa1F, VP16, or a mammaliannuclear receptor. In another embodiment of the rescue screen, at leastone of the heterologous fusion proteins is expressed from anautonomously-replicating plasmid. In another embodiment, the DNA bindingdomain is a heterologous DNA-binding domain of a transcriptionalactivation protein.

[0109] The invention also provides for a method for identifyingpotential compounds which modulate Dkk activity comprising:

[0110] a) measuring the effect on binding of one or more Dkk interactingprotein, or a Dkk-binding fragment thereof, with Dkk or a LRP5/LRP6/HBMbinding fragment thereof in the presence and absence of a compound; and

[0111] b) identifying as a potential Dkk modulatory compound a compoundwhich modulates the binding between one or more Dkk interacting proteinsor Dkk-binding fragment thereof and Dkk or LRP5/LRP6/HBM fragmentthereof.

[0112] In a preferred embodiment, the Dkk is Dkk-1.

[0113] The invention further provides for any of the Dkk peptideaptamers of FIG. 3 (SEQ ID NOs:171-188). The invention also provides forany of the LRP peptide aptamers of FIG. 4 (SEQ ID NOs:189-192).

[0114] Another aspect of the invention provides for a method ofidentifying agents which modulate the interaction of Dkk with the Wntsignaling pathway comprising:

[0115] (a) injecting mRNA encoding Dkk and an agent into a Xenopusblastomere;

[0116] (b) assessing axis duplication or analyzing marker geneexpression; and

[0117] (c) identifying agents which elicit changes in axis duplicationor marker gene expression as agents which modulate the interaction ofDkk with the Wnt signaling pathway. Wherein the agent may be chosen fromamong mRNA encoding Dkk interacting proteins, fragments thereof, siRNA,shRNA, antisense nucleotides, and antibodies. In a preferred embodiment,Dkk is Dkk-1. In a further embodiment, mRNA of HBM, LRP5/6, any Wnt(including Wnt1-Wnt19, particularly Wnt1, Wnt3, Wnt3a, and Wnt10b), Wntantagonist, or combination of these is co-injected into the Xenopusblastomere. In another embodiment, the marker gene analyzed couldinclude Siamois, Xnr3, slug, Xbra, HNK-1, endodermin, Xlhbox8, BMP2,BMP4, XLRP6, EF-1, or ODC.

[0118] The present invention provides for a method for identifyingagents which modulate the interaction of Dkk with the Wnt signalingpathway comprising:

[0119] (a) transfecting cells with constructs encoding Dkk and potentialDkk interacting proteins, mRNA fragments thereof, siRNA, shRNA, orantisense, antibodies to LRP5/HBM/LRP6/Dkk/Dkk-interacting protein;

[0120] (b) assessing changes in expression of a reporter gene linked toa Wnt-responsive promoter; and,

[0121] (c) identifying as a Dkk interacting protein any protein whichalters reporter gene expression compared with cells transfected with aDkk construct alone. In a further preferred embodiment, the cells may beHOB-03-CE6, HEK293, or U2OS cells.

[0122] In alternative embodiments, the Wnt-responsive promoter is TCF orLEF. In other preferred embodiments, the cells are co-transfected withCMV beta-galactosidase or tk-Renilla.

[0123] The present invention further provides for a LRP5/HBM monoclonalor polyclonal antibody to one or more peptides of amino acid sequencesMYWTDWVETPRIE, (SEQ ID NO:123) MYWTDWGETPRIE, (SEQ ID NO:124)KRTGGKRKEILSA, (SEQ ID NO:125) ERVEKTTGDKRTRIQGR, (SEQ ID NO:126) orKQQCDSFPDCIDGSDE. (SEQ ID NO:127)

[0124] Additionally, the present invention provides a method foridentifying compounds which modulate Dkk and LRP5/LRP6/HBM interactionscomprising:

[0125] (a) immobilizing LRP5/LRP6/HBM to a solid surface; and

[0126] (b) treating the solid surface with a secreted Dkk protein or asecreted epitope-tagged Dkk and a test compound; and

[0127] (c) determining whether the compound regulates binding betweenDkk and LRP5/LRP6/HMB using antibodies to Dkk or the epitope tag or bydirectly measuring activity of an epitope tag.

[0128] In one embodiment, the Dkk is Dkk-1. In a preferred embodiment,the epitope tag is alkaline phosphatase, histidine, myc, or a V5 tag.

[0129] Another embodiment of the present invention provides for a methodfor identifying compounds which modulate Dkk and LRP5/LRP6/HBMinteractions comprising:

[0130] (a) creating an LRP5, LRP6, or HBM fluorescent fusion proteinusing a first fluorescent tag;

[0131] (b) creating a Dkk fusion protein comprising a second fluorescenttag;

[0132] (c) adding a test compound; and,

[0133] (d) assessing changes in the ratio of fluorescent tag emissionsusing Fluorescence Resonance Energy Transfer (FRET) or BioluminescentResonance Energy Transfer (BRET) to determine whether the compoundmodulates Dkk and LRP5/LRP6/HBM interactions.

[0134] In a preferred embodiment, the Dkk is Dkk-1.

[0135] The present invention also provides for a method of diagnosinglow or high bone mass and/or low or high lipid levels in a subjectcomprising examining expression of Dkk, LRP5, LRP6, HBM or HBM-likevariant in the subject and determining whether Dkk, LRP5, LRP6, or HBMor a HBM-like variant is over- or under-expressed to determine whethersubject has (a) high or low bone mass and/or (b) high or low lipidlevels.

[0136] The invention further provides for a transgenic animal whereinDkk is knocked out in a tissue-specific fashion. In a preferredembodiment, the Dkk is Dkk-1. In one preferred embodiment, the tissuespecificity is bone tissue. In another preferred embodiment, the tissuespecificity is liver or other tissues or cells involved in regulatinglipid metabolism or cancer tissue.

[0137] The present invention further provides a method of screening forcompounds which modulate the interaction of Dkk with LRP5, LRP6, or HBMcomprising:

[0138] (a) exposing LRP5, LRP6, or HBM, or a Dkk-binding fragment ofLRP5, LRP6, or HBM to a compound; and

[0139] (b) determining whether said compound bound to LRP5, LRP6, or HBMor the Dkk-binding fragment of LRP5, LRP6, or HBM and furtherdetermining whether said compound modulates the interaction of Dkk andLRP5, LRP6, or HBM.

[0140] In one embodiment, the Dkk is Dkk-1. In a preferred embodiment,the compound comprises an LRP5 peptide aptamer. Other preferredcompositions include the peptide aptamer, OST262 (SEQ ID NO:208), FIG. 4(SEQ ID NOs:189-192) (particularly peptide 13 (SEQ ID NO:191) and FIG.13 (including SEQ ID NOs:204-214), or a mimetic of such an aptamer, andan LRP5 antibody.

[0141] The present invention also provides a method for identifyingcompounds which modulate Dkk and LRP5/LRP6/HBM interactions comprising:

[0142] (a) immobilizing LRP5/LRP6/HBM to a solid surface; and

[0143] (b) treating the solid surface with a secreted Dkk protein or asecreted epitope-tagged Dkk and a test compound; and

[0144] (c) determining whether the compound regulates binding betweenDkk and LRP5/LRP6/HBM using antibodies to Dkk or the epitope tag or bydirectly measuring activity of an epitope tag. In a preferredembodiment, the epitope tag is alkaline phosphatase, histidine, myc or aV5 tag.

[0145] In a preferred embodiment, the Dkk is Dkk-1.

[0146] The invention also provides for a method for identifyingcompounds which modulate the interaction of Dkk with the Wnt signalingpathway comprising:

[0147] (a) transfecting cells with constructs containing Dkk and Wntproteins;

[0148] (b) assessing changes in expression of a reporter element linkedto a Wnt-responsive promoter; and

[0149] (c) identifying as a Dkk/Wnt interaction modulating compound anycompound which alters reporter gene expression compared with cellstransfected with a Dkk construct alone.

[0150] In one embodiment, the Dkk is Dkk-1. In another embodiment, theWnt is any of Wnt1-Wnt19. In a preferred embodiment, the Wnt is Wnt1,Wnt3, Wnt3a, or Wnt10b. In a particularly preferred embodiment, the Wntconstruct contains Wnt3a. In another particularly preferred embodiment,the Wnt construct contains Wnt1. In another preferred embodiment, theWnt construct encodes for a Wnt that signals through the canonical Wntpathway. In a particularly preferred embodiment, both Wnt3a and Wnt1constructs are co-transfected into the cells. In another embodiment, thecells may be U2-OS, HOB-03-CE6, or HEK293 cells. In another embodiment,the reporter element used is TCF-luciferase, tk-Renilla, or acombination thereof.

[0151] The invention also provides for a method of testing compoundsthat modulate Dkk-mediated activity in a mammal comprising:

[0152] (a) providing a group of transgenic animals having (1) aregulatable one or more Dkk genes, (2) a knock-out of Dkk genes, or (3)a knock-in of one or more Dkk genes;

[0153] (b) providing a second group of control animals respectively forthe group of transgenic animals in step (a); and

[0154] (c) exposing the transgenic animal group and control animal groupto a potential Dkk-modulating compound which modulates bone mass orlipid levels; and

[0155] (d) comparing the transgenic animals and the control group ofanimals and determining the effect of the compound on bone mass or lipidlevels in the transgenic animals as compared to the control animals.

[0156] In a preferred embodiment, the Dkk is Dkk-1.

[0157] The invention further provides variants of LRP5 which demonstrateHBM biological activity, i.e., that are “HBM-like.” In preferredembodiments, variants G171F, M282V, G171K, G171Q, A65V, G171V, G171I,and A214V of LRP5 are provided. The invention further provides for theuse any of these variants in the forgoing methods.

BRIEF DESCRIPTION OF THE FIGURES

[0158]FIG. 1 shows a schematic of the components of the Wnt signaltransduction pathway. Schematic obtained from:http://www.stanford.edu/˜rnusse/pathways/cell2.html

[0159]FIG. 2 (A-C) show bait sequences (SEQ ID NOs:168-170) utilized inyeast two hybrid (Y2H) screens for protein-protein interactions.

[0160]FIG. 3 shows a table of peptide aptamer insert sequences (SEQ IDNOs: 171-192) identified in Y2H screen with a Dkk-1 bait sequence.

[0161]FIG. 4 shows a table of peptide aptamer insert sequencesidentified in a Y2H screen using a LRP5 ligand binding domain baitsequence.

[0162]FIG. 5 shows a table of proteins identified in a Y2H screen usinga Dkk-1 bait sequence. These proteins are identified by both theirnucleic acid and amino acid accession numbers.

[0163]FIG. 6 shows the results of a minimum interaction domain mappingscreen of Dkk-1 with LRP5. At the top, a map of Dkk-1 showing thelocation of the signal sequence, and cysteine rich domains 1 and 2.Below, the extent of domains examined using LRP5 LBD baits, LBD1 andLBD4, of FIG. 2. To the right, scoring of the binding results observedin the experiment.

[0164]FIG. 7 shows a diagram of the Xenopus Embryo Assay for Wntactivity.

[0165]FIG. 8 shows the effects of Zmax/LRP5 and HBM on Wnt signaling inthe Xenopus embryo assay.

[0166]FIG. 9 shows the effects of Zmax/LRP5 and HBM on induction ofsecondary axis formation in the Xenopus embryo assay.

[0167]FIG. 10 shows the effects of human Dkk-1 on the repression of thecanonical Wnt pathway.

[0168]FIG. 11 shows the effects of human Dkk-1 on Zmax/LRP5 andHBM-mediated Wnt signaling.

[0169]FIG. 12 shows pcDNA3.1 construct names with nucleotide sequences(including SEQ ID NOs:193-203) for LRP5-binding peptide aptamers, Dkk-1peptides and control constructs.

[0170]FIG. 13 shows the amino acid sequences (including SEQ IDNOs:204-214) for the corresponding LRP5-binding peptides, Dkk-1 peptideaptamers and control constructs in FIG. 12.

[0171]FIG. 14 shows the effects of Dkk-1 and Dkk-2 on Wnt1 signalingwith coreceptors LRP5, HBM, and LRP6 in HOB03CE6 cells.

[0172]FIG. 15 shows the effects of Dkk-1 and Dkk-2 on Wnt3a signalingwith coreceptors LRP5, HBM, and LRP6 in HOB03CE6 cells.

[0173]FIG. 16 demonstrates that the LRP5-LBD peptide aptamer 262activates Wnt signaling in the presence of Wnt3a in U2OS cells.

[0174]FIG. 17 shows the differential binding of an antibody generated toa sequence (a.a. 165-177) containing the HBM mutation in LRP5 in LRP5and HBM virus-infected cells.

[0175]FIG. 18 shows data generated from a Y2H interaction trap where amutant Dkk-1 (C220A) is unable to bind to LRP5 and demonstrating thewindow of capability of detecting small molecule effects on LRP and Dkkinteractions.

[0176]FIG. 19 shows that Dkk-1 represses Wnt3a-mediated Wnt signaling inU2OS bone cells using the cell-based reporter gene assay for highthroughput screening.

[0177]FIG. 20 demonstrates that Wnt1-HBM generated signaling is notefficiently inhibited by Dkk-1 in U2OS bone cells while LRP5 andLRP6-mediated signaling are using the cell-based reporter gene assay forhigh throughput screening.

[0178]FIG. 21 shows that the TCF signal in the cell-based reporter geneassay for high throughput screening can be modulated by Dkk-1 andDkk-1-AP without Wnt DNA transfection.

[0179]FIG. 22 shows the morphological results in the Xenopus assay usingaptamers 261 and 262 from the LRP5-LBD to activate Wnt signaling.

[0180]FIG. 23 demonstrates that LRP5-LBD aptamers 261 and 262 induce Wntsignaling over other LRP5 aptamers.

[0181]FIG. 24 shows that the mutation G171 F in LRP5 produces a greateractivation of the Wnt pathway than LRP5 which is consistent with HBMactivity.

[0182]FIG. 25 shows that the mutation M282V in LRP5 produces anactivation of the Wnt pathway which is consistent with HBM activity inU2OS cells.

[0183]FIG. 26 shows the amino acid sequence of the various peptides ofdkk-1 selected to generate polyclonal antibodies, their relationship tothe Dkk-1 amino acid sequence and identities of polyclonal antibodiesgenerated.

[0184]FIG. 27 shows a Western blot demonstrating that polyclonalantibody #5521 to amino acids 165-186 of Dkk-1 was able to detect Dkk1-V5 and Dkk1-AP from conditioned medium.

[0185]FIG. 28 shows a Western blot demonstrating that polyclonalantibody #74397 to amino acids 147-161 was able to detect Dkk1-V5 inboth conditioned medium and immunoprecipitated conditioned medium.

DETAILED DESCRIPTION OF THE INVENTION

[0186] 1. Definitions

[0187] In general, terms in the present application are used consistentwith the manner in which those terms are understood in the art. To aidin the understanding of the specification and claims, the followingdefinitions are provided.

[0188] “Gene” refers to a DNA sequence that encodes through its templateor messenger RNA a sequence of amino acids characteristic of a specificpeptide. The term “gene” includes intervening, non-coding regions, aswell as regulatory regions, and can include 5′ and 3′ ends.

[0189] By “nucleic acid” is meant to include single stranded and doublestranded nucleic acids including, but not limited to DNAs, RNAs (e.g.,mRNA, tRNAs, siRNAs), cDNAs, recombinant DNA (rDNA), rRNAs, antisensenucleic acids, oligonucleotides, and oligomers, and polynucleotides. Theterm may also include hybrids such as triple stranded regions of RNAand/or DNA or double stranded RNA:DNA hybrids. The term also iscontemplated to include modified nucleic acids such as, but not limitedto biotinylated nucleic acids, tritylated nucleic acids, fluorophorlabeled nucleic acids, inosine, and the like.

[0190] “Gene sequence” refers to a nucleic acid molecule, including DNAwhich contains a non-transcribed or non-translated sequence, whichcomprises a gene. The term is also intended to include any combinationof gene(s), gene fragment(s), non-transcribed sequence(s) ornon-translated sequence(s) which are present on the same DNA molecule.

[0191] The nucleic acid sequences of the present invention may bederived from a variety of sources including DNA, cDNA, synthetic DNA,synthetic RNA or combinations thereof. Such sequences may comprisegenomic DNA which may or may not include naturally occurring introns.Moreover, such genomic DNA may be obtained in association with promoterregions and/or poly (A) sequences. The sequences, genomic DNA or cDNAmay be obtained in any of several ways. Genomic DNA can be extracted andpurified from suitable cells by means well known in the art.Alternatively, mRNA can be isolated from a cell and used to produce cDNAby reverse transcription or other means.

[0192] “cDNA” refers to complementary or copy DNA produced from an RNAtemplate by the action of RNA-dependent DNA polymerase (reversetranscriptase). Thus, a “cDNA clone” means a duplex DNA sequence forwhich one strand is complementary to an RNA molecule of interest,carried in a cloning vector or PCR amplified. cDNA can also be singlestranded after first strand synthesis by reverse transcriptase. In thisform, it is a useful PCR template and does not need to be carried in acloning vector. This term includes genes from which the interveningsequences have been removed. Thus, the term “gene”, as sometimes usedgenerically, can also include nucleic acid molecules comprising cDNA andcDNA clones.

[0193] “Recombinant DNA” means a molecule that has been engineered bysplicing in vitro a cDNA or genomic DNA sequence or altering a sequenceby methods such as PCR mutagenesis.

[0194] “Cloning” refers to the use of in vitro recombination techniquesto insert a particular gene or other DNA sequence into a vectormolecule. In order to successfully clone a desired gene, it is necessaryto use methods for generating DNA fragments, for joining the fragmentsto vector molecules, for introducing the composite DNA molecule into ahost cell in which it can replicate, and for selecting the clone havingthe target gene from amongst the recipient host cells.

[0195] “cDNA library” refers to a collection of recombinant DNAmolecules containing cDNA inserts which together comprise the entire ora partial repertoire of genes expressed in a particular tissue or cellsource. Such a cDNA library can be prepared by methods known to oneskilled in the art and described by, for example, Cowell and Austin,“cDNA Library Protocols,” Methods in Molecular Biology (1997).

[0196] “Cloning vehicle” refers to a plasmid or phage DNA or other DNAsequence which is able to replicate in a host cell. This term can alsoinclude artificial chromosomes such as BACs and YACs. The cloningvehicle is characterized by one or more endonuclease recognition sitesat which such DNA sequences may be cut in a determinable fashion withoutloss of an essential biological function of the DNA, which may contain amarker suitable for use in the identification of transformed cells.

[0197] “Expression” refers to the process comprising transcription of agene sequence and subsequent processing steps, such as translation of aresultant mRNA to produce the final end product of a gene. The endproduct may be a protein (such as an enzyme or receptor) or a nucleicacid (such as a tRNA, antisense RNA, or other regulatory factor). Theterm “expression control sequence” refers to a sequence of nucleotidesthat control or regulate expression of structural genes when operablylinked to those genes. These include, for example, the lac systems, thetrp system, major operator and promoter regions of the phage lambda, thecontrol region of fd coat protein and other sequences known to controlthe expression of genes in prokaryotic or eukaryotic cells. Expressioncontrol sequences will vary depending on whether the vector is designedto express the operably linked gene in a prokaryotic or eukaryotic host,and may contain transcriptional elements such as enhancer elements,termination sequences, tissue-specificity elements and/or translationalinitiation and termination sites.

[0198] “Expression vehicle” refers to a vehicle or vector similar to acloning vehicle but which is capable of expressing a gene which has beencloned into it, after transformation into a host. The cloned gene isusually placed under the control of (i.e., operably linked to) anexpression control sequence.

[0199] “Operator” refers to a DNA sequence capable of interacting withthe specific repressor, thereby controlling the transcription ofadjacent gene(s).

[0200] “Promoter” refers to a DNA sequence that can be recognized by anRNA polymerase. The presence of such a sequence permits the RNApolymerase to bind and initiate transcription of operably linked genesequences.

[0201] “Promoter region” is intended to include the promoter as well asother gene sequences which may be necessary for the initiation oftranscription. The presence of a promoter region is sufficient to causethe expression of an operably linked gene sequence. The term “promoter”is sometimes used in the art to generically indicate a promoter region.Many different promoters are known in the art which direct expression ofa gene in a certain cell types. Tissue-specific promoters can comprisenucleic acid sequences which cause a greater (or decreased) level ofexpression in cells of a certain tissue type.

[0202] “Operably linked” means that the promoter controls the initiationof expression of the gene. A promoter is operably linked to a sequenceof proximal DNA if upon introduction into a host cell the promoterdetermines the transcription of the proximal DNA sequence(s) into one ormore species of RNA. A promoter is operably linked to a DNA sequence ifthe promoter is capable of initiating transcription of that DNAsequence.

[0203] “Prokaryote” refers to all organisms without a true nucleus,including bacteria.

[0204] “Eukaryote” refers to organisms and cells that have a truenucleus, including mammalian cells.

[0205] “Host” includes prokaryotes and eukaryotes, such as yeast andfilamentous fungi, as well as plant and animal cells. The term includesan organism or cell that is the recipient of a replicable expressionvehicle.

[0206] The term “animal” is used herein to include all vertebrateanimals, except humans. It also includes an individual animal in allstages of development, including embryonic and fetal stages. Preferredanimals include higher eukaryotes such as avians, rodents (e.g., mice,rabbits, rats, chinchillas, guinea pigs, hamsters and the like), andmammals. Preferred mammals include bovine, equine, feline, canine,ovine, caprine, porcine, buffalo, humans, and primates.

[0207] A “transgenic animal” is an animal containing one or more cellsbearing genetic information received, directly or indirectly, bydeliberate genetic manipulation or by inheritance from a manipulatedprogenitor at a subcellular level, such as by microinjection orinfection with a recombinant viral vector (e.g., adenovirus, retrovirus,herpes virus, adeno-associated virus, lentivirus). This introduced DNAmolecule may be integrated within a chromosome, or it may beextra-chromosomally replicating DNA.

[0208] “Embryonic stem cells” or “ES cells” as used herein are cells orcell lines usually derived from embryos which are pluripotent meaningthat they are undifferentiated cells. These cells are also capable ofincorporating exogenous DNA by homologous recombination and subsequentlydeveloping into any tissue in the body when incorporated into a hostembryo. It is possible to isolate pluripotent cells from sources otherthan embryonic tissue by methods which are well understood in the art.

[0209] Embryonic stem cells in mice have enabled researchers to selectfor transgenic cells and perform gene targeting. This allows moregenetic engineering than is possible with other transgenic techniques.For example, mouse ES cells are relatively easy to grow as colonies invitro. The cells can be transfected by standard procedures andtransgenic cells clonally selected by antibiotic resistance. See, forexample, Doetschman et al., 1994, Gene transfer in embryonic stem cells.In Pinkert (Ed.) Transgenic Animal Technology: A Laboratory Handbook.Academic Press Inc., New York, pp.115-146. Furthermore, the efficiencyof this process is such that sufficient transgenic colonies (hundreds tothousands) can be produced to allow a second selection for homologousrecombinants. Mouse ES cells can then be combined with a normal hostembryo and, because they retain their potency, can develop into all thetissues in the resulting chimeric animal, including the germ cells. Thetransgenic modification can then be transmitted to subsequentgenerations.

[0210] Methods for deriving embryonic stem (ES) cell lines in vitro fromearly preimplantation mouse embryos are well known. See for example,Evans et al., 1981 Nature 29:154-6 and Martin, 1981, Proc. Nat. Acad.Sci. USA, 78: 7634-8. ES cells can be passaged in an undifferentiatedstate, provided that a feeder layer of fibroblast cells or adifferentiation inhibiting source is present.

[0211] The term “somatic cell” indicates any animal or human cell whichis not a sperm or egg cell or is capable of becoming a sperm or eggcell. The term “germ cell” or “germ-line cell” refers to any cell whichis either a sperm or egg cell or is capable of developing into a spermor egg cell and can therefore pass its genetic information to offspring.The term “germ cell-line transgenic animal” refers to a transgenicanimal in which the genetic information was incorporated in a germ linecell, thereby conferring the ability to transfer the information tooffspring. If such offspring in fact possess some or all of thatinformation, then they, too, are transgenic animals.

[0212] The genetic alteration of genetic information may be foreign tothe species of animal to which the recipient belongs, or foreign only tothe particular individual recipient. In the last case, the altered orintroduced gene may be expressed differently than the native gene.

[0213] “Fragment” of a gene refers to any portion of a gene sequence. A“biologically active fragment” refers to any portion of the gene thatretains at least one biological activity of that gene. For example, thefragment can perhaps hybridize to its cognate sequence or is capable ofbeing translated into a polypeptide fragment encoded by the gene fromwhich it is derived.

[0214] “Variant” refers to a gene that is substantially similar instructure and biological activity or immunological characteristics toeither the entire gene or to a fragment of the gene. Provided that thetwo genes possess a similar activity, they are considered variant asthat term is used herein even if the sequence of encoded amino acidresidues is not identical. Preferentially, as used herein (unlessotherwise defined) the variant is one of LRP5, HBM or LRP6. The variantpreferably is one that yields an HBM-like phenotype (i.e., enhancesbones mass and/or modulates lipid levels). These variants includemissense mutations, single nucleotide polymorphisms (SNPs), mutationswhich result in changes in the amino acid sequence of the proteinencoded by the gene or nucleic acid, and combinations thereof, as wellas corn in the exon domains of the HBM gene and mutations in LRP5 orLRP6 which result in an HBM like phenotype.

[0215] “Amplification of nucleic acids” refers to methods such aspolymerase chain reaction (PCR), ligation amplification (or ligase chainreaction, LCR) and amplification methods based on the use of Q-betareplicase. These methods are well known in the art and described, forexample, in U.S. Pat. Nos. 4,683,195 and 4,683,202. Reagents andhardware for conducting PCR are commercially available. Primers usefulfor amplifying sequences from the HBM region are preferablycomplementary to, and hybridize specifically to sequences in the HBMregion or in regions that flank a target region therein. HBM sequencesgenerated by amplification may be sequenced directly. Alternatively, theamplified sequence(s) may be cloned prior to sequence analysis.

[0216] “Antibodies” may refer to polyclonal and/or monoclonal antibodiesand fragments thereof, and immunologic binding equivalents thereof, thatcan bind to the HBM proteins and fragments thereof or to nucleic acidsequences from the HBM region, particularly from the HBM locus or aportion thereof. Preferred antibodies also include those capable ofbinding to LRP5, LRP6 and HBM variants. The term antibody is used bothto refer to a homogeneous molecular entity, or a mixture such as a serumproduct made up of a plurality of different molecular entities. Proteinsmay be prepared synthetically in a protein synthesizer and coupled to acarrier molecule and injected over several months into rabbits. Rabbitsera is tested for immunoreactivity to the HBM protein or fragment.Monoclonal antibodies may be made by injecting mice with the proteins,or fragments thereof. Monoclonal antibodies will be screened by ELISAand tested for specific immunoreactivity with HBM protein or fragmentsthereof. Harlow et al., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1988) and Using Antibodies:A Laboratory Manual, Harlow, Ed and Lane, David (Cold Spring HarborPress, 1999). These antibodies will be useful in assays as well aspharmaceuticals. By “antibody” is meant to include but not limited topolyclonal, monoclonal, chimeric-, human, humanized, bispecific,multispecific, primatized™ antibodies.

[0217] “HBM protein” refers to a protein that is identical to a Zmax1(LRP5) protein except that it contains an alteration of glycine 171 to avaline. An HBM protein is defined for any organism that encodes a Zmax1(LRP5) true homolog. For example, a mouse HBM protein refers to themouse Zmax1 (LRP5) protein having the glycine 170 to valinesubstitution.

[0218] By “HBM-like” is meant a variant of LRP5, LRP6 or HBM which whenexpressed in a cell is capable of modulating bone mass, lipid levels,Dkk activity, and/or Wnt activity.

[0219] In one embodiment of the present invention, “HBM gene” refers tothe genomic DNA sequence found in individuals showing the HBMcharacteristic or phenotype, where the sequence encodes the proteinindicated by SEQ ID NO: 4. The HBM gene and the Zmax1 (LRP5) gene areallelic. The protein encoded by the HBM gene has the property of causingelevated bone mass, while the protein encoded by the Zmax1 (LRP5) genedoes not. The HBM gene and the Zmax1 (LRP5) gene differ in that the HBMgene has a thymine at position 582, while the Zmax1 gene has a guanineat position 582. The HBM gene comprises the nucleic acid sequence shownas SEQ ID NO: 2. The HBM gene may also be referred to as an “HBMpolymorphism.” Other HBM genes may further have silent mutations, suchas those discussed in Section 3 below.

[0220] In alternative embodiments of the present invention, “HBM gene”may also refer to any allelic variant of Zmax1 (LRP5) or LRP6 whichresults in the HBM phenotype. Such variants may include alteration fromthe wild-type protein coding sequence as described herein and/oralteration in expression control sequences of Zmax1 (LRP5) or containsan amino acid mutation in LRP5 or LRP6, such that the resulting proteinproduces a phenotype which enhances bone mass and/or modulates lipidlevels. A preferred example of such a variant is an alteration of theendogenous Zmax1 (LRP5) promoter region resulting in increasedexpression of the Zmax1 (LRP5) protein.

[0221] “Normal,” “wild-type,” “unaffected”, “Zmax1”, “Zmax”, “LR3” and“LRP5” all refer to the genomic DNA sequence that encodes the proteinindicated by SEQ ID NO: 3. LRP5 has also been referred to LRP7 in mouse.Zmax1, LRP5 and Zmax may be used interchangeably throughout thespecification and are meant to be the same gene, perhaps only relatingto the gene in a different organism. The Zmax1 gene has a guanine atposition 582 in the human sequence. The Zmax1 gene of human comprisesthe nucleic acid sequence shown as SEQ ID NO: 1. “Normal,” “wild-type,”“unaffected”, “Zmax1” and “LRP5” also refer to allelic variants of thegenomic sequence that encodes proteins that do not contribute toelevated bone mass. The Zmax1 (LRP5) gene is common in the humanpopulation, while the HBM gene is rare.

[0222] “Bone development” generally refers to any process involved inthe change of bone over time, including, for example, normaldevelopment, changes that occur during disease states, and changes thatoccur during aging. This may refer to structural changes and dynamicrate changes such as growth rates, resorption rates, bone repair rates,and etc. “Bone development disorder” particularly refers to anydisorders in bone development including, for example, changes that occurduring disease states and changes that occur during aging. Bonedevelopment may be progressive or cyclical in nature. Aspects of bonethat may change during development include, for example, mineralization,formation of specific anatomical features, and relative or absolutenumbers of various cell types.

[0223] “Bone modulation” or “modulation of bone formation” refers to theability to affect any of the physiological processes involved in boneremodeling, as will be appreciated by one skilled in the art, including,for example, bone resorption and appositional bone growth, by, interalia, osteoclastic and osteoblastic activity, and may comprise some orall of bone formation and development as used herein.

[0224] Bone is a dynamic tissue that is continually adapting andrenewing itself through the renewal of old or unnecessary bone byosteoclasts and the rebuilding of new bone by osteoblasts. The nature ofthe coupling between these processes is responsible for both themodeling of bone during growth as well as the maintenance of adultskeletal integrity through remodeling and repair to meet the everydayneeds of mechanical usage. There are a number of diseases that resultfrom an uncoupling of the balance between bone resorption and formation.With aging there is a gradual “physiologic” imbalance in bone turnover,which is particularly exacerbated in women due to menopausal loss ofestrogen support, that leads to a progressive loss of bone. As bonemineral density falls below population norms there is a consequentincrease in bone fragility and susceptibility to spontaneous fractures.For every 10 percent of bone that is lost, the risk of fracture doubles.Individuals with bone mineral density (BMD) in the spine or proximalfemur 2.5 or more standard deviations below normal peak bone mass areclassified as osteoporotic. However, osteopenic individuals with BMDbetween 1 and 2.5 standard deviations below the norm are clearly atrisk.

[0225] Bone is measured by several different forms of X-rayabsorptiometry. All of the instruments measure the inorganic or bonemineral content of the bone. Standard DXA measurements give a value thatis an areal density, not a true density measurement by the classicaldefinition of density (mass/unit volume). Nevertheless, this is the typeof measurement used clinically to diagnose osteoporosis. However, whileBMD is a major contributing factor to bone strength, as much as 40% ofbone strength stems from other factors including: 1) bone size (i.e.,larger diameters increase organ-level stiffness, even in the face oflower density); 2) the connectivity of trabecular structures; 3) thelevel of remodeling (remodeling loci are local concentrators of strain);and 4) the intrinsic strength of the bony material itself, which in turnis a function of loading history (i.e., through accumulated fatiguedamage) and the extent of collagen cross-linking and level ofmineralization. There is good evidence that all of thesestrength/fragility factors play some role in osteoporotic fractures, asdo a host of extraskeletal influences as well (such as fall patterns,soft tissue padding, and central nervous system reflex responsiveness).

[0226] Additional analytical instruments can be used to address thesefeatures of bone. For example, the PQCT allows measurement of separatetrabecular and cortical compartments for size and density and the μCTprovides quantitative information on architectural features such astrabecular connectivity. The μCT also gives a true bone densitymeasurement. With these tools, the important non-BMD parameters can bemeasured for diagnosing the extent of disease and the efficacy oftreatments. Current treatments for osteoporosis are based on the abilityof drugs to prevent or retard bone resorption. Although neweranti-resorptive agents are proving to be useful in the therapy ofosteoporosis, they are viewed as short-term solutions to the moredefinitive challenge to develop treatments that will increase bone massand/or the bone quality parameters mentioned above.

[0227] Thus, bone modulation may be assessed by measuring parameterssuch as bone mineral density (BMD) and bone mineral content (BMC) bypDXA X-ray methods, bone size, thickness or volume as measured by X-ray,bone formation rates as measured for example by calcien labeling, total,trabecular, and mid-shaft density as measured by pQCT and/or μCTmethods, connectivity and other histological parameters as measured byμCT methods, mechanical bending and compressive strengths as preferablymeasured in femur and vertebrae respectively. Due to the nature of thesemeasurements, each may be more or less appropriate for a given situationas the skilled practitioner will appreciate. Furthermore, parameters andmethodologies such as a clinical history of freedom from fracture, boneshape, bone morphology, connectivity, normal histology, fracture repairrates, and other bone quality parameters are known and used in the art.Most preferably, bone quality may be assessed by the compressivestrength of vertebra when such a measurement is appropriate. Bonemodulation may also be assessed by rates of change in the variousparameters. Most preferably, bone modulation is assessed at more thanone age.

[0228] “Normal bone density” refers to a bone density within twostandard deviations of a Z score of 0 in the context of the HBM linkagestudy. In a general context, the range of normal bone density parametersis determined by routine statistical methods. A normal parameter iswithin about 1 or 2 standard deviations of the age and sex normalizedparameter, preferably about 2 standard deviations. A statistical measureof meaningfulness is the P value which can represent the likelihood thatthe associated measurement is significantly different from the mean.Significant P values are P<0.05, 0.01, 0.005, and 0.001, preferably atleast P<0.01.

[0229] “HBM” refers to “high bone mass” although this term may also beexpressed in terms of bone density, mineral content, and size.

[0230] The “HBM phenotype” and “HBM-like phenotype” may be characterizedby an increase of about 2 or more standard deviations, preferably 2,2.5, 3, or more standard deviations in 1, 2, 3, 4, 5, or morequantitative parameters of bone modulation, preferably bone density andmineral content and bone strength parameters, above the age and sex normfor that parameter. The HBM phenotype and HBM-like phenotype arecharacterized by statistically significant increases in at least oneparameter, preferably at least 2 parameters, and more preferably atleast 3 or more parameters. The HBM phenotype and the HBM-like phenotypemay also be characterized by an increase in one or more bone qualityparameters and most preferably increasing parameters are not accompaniedby a decrease in any bone quality parameters. Most preferably, anincrease in bone modulation parameters and/or bone quality measurementsis observed at more than one age. The HBM phenotype and HBM-likephenotype also includes changes of lipid levels, Wnt activity and/or Dkkactivity.

[0231] The terms “isolated” and “purified” refer to a substance alteredby hand of man from the natural environment. An isolated peptide may befor example in a substantially pure form or otherwise displaced from itsnative environment such as by expression in an isolated cell line ortransgenic animal. An isolated sequence may for example be a molecule insubstantially pure form or displaced from its native environment suchthat at least one end of said isolated sequence is not contiguous withthe sequence it would be contiguous with in nature.

[0232] “Biologically active” refers to those forms of proteins andpolypeptides, including conservatively substituted variants, alleles ofgenes encoding a protein or polypeptide fragments of proteins whichretain a biological and/or immunological activity of the wild-typeprotein or polypeptide. Preferably the activity is one which induces achange in Dkk activity, such as inhibiting the interaction of Dkk with aligand binding partner (e.g., LRP5 or LRP6 or Dkk-1 with a Dkk-1interacting protein such as those shown in FIG. 5). By biologicallyactive is also meant to include any form which modulates Wnt signaling.

[0233] By “modulate” and “regulate” is meant methods, conditions, oragents which increase or decrease the wild-type activity of an enzyme,inhibitor, signal transducer, receptor, transcription activator,co-factor, and the like. This change in activity can be an increase ordecrease of mRNA translation, mRNA or DNA transcription, and/or mRNA orprotein degradation, which may in turn correspond to an increase ordecrease in biological activity.

[0234] By “modulated activity” is meant any activity, condition, diseaseor phenotype which is modulated by a biologically active form of aprotein. Modulation may be effected by affecting the concentration orsubcellular localization of biologically active protein, i.e., byregulating expression or degradation, or by direct agonistic orantagonistic effect as, for example, through inhibition, activation,binding, or release of substrate, modification either chemically orstructurally, or by direct or indirect interaction which may involveadditional factors.

[0235] By “effective amount” or “dose effective amount” or“therapeutically effective amount” is meant an amount of an agent whichmodulates a biological activity of the polypeptide of the invention.

[0236] By “immunologically active” is meant any immunoglobulin proteinor fragment thereof which recognizes and binds to an antigen.

[0237] By “Dkk” is meant to refer to the nucleic acids and proteins ofmembers of the Dkk (Dickkopf) family. This includes, but is not limitedto, Dkk-1, Dkk-2, Dkk-3, Dkk-4, Soggy, and related Dkk proteins. Dkk-1is a preferred embodiment of the present invention. However, the Dkkproteins have substantial homology and one skilled in the art willappreciate that all of the embodiments of the present inventionutilizing Dkk-1 may also be utilized with the other Dkk proteins.

[0238] By “Dkk-1” is meant to refer to the Dkk-1 protein and nucleicacids which encode the Dkk-1 protein. Dkk-1 refers to Dickkopf-1, and inXenopus it is related to at least Dkk-2, Dkk-3, and Dkk-4 (see Krupniket al., Gene 238:301-313 (1999)). Dkk-1 was first identified in Xenopus(Glinka et al., Nature 391:357-62 (1998)). It was recognized as a factorcapable of inducing ectopic head formation in the presence of inhibitionof the BMP pathway. It was then also found to inhibit the axis-inducingactivity of several Xenopus Wnt molecules by acting as an extracellularantagonist of Wnt signaling. Mammalian homologs have been foundincluding Dkk-1, Dkk-2, Dkk-3, Dkk-4 and soggy (Fedi et al., 1999 andKrupnick et al. 1999). Human Dkk-1 was also referred to as sk (Fedi etal. 1999). As used herein, Dkk-1 is meant to include proteins from anyspecies having a Wnt pathway in which Dkk-1 interacts. Particularlypreferred are mammalian species (e.g., murine, caprine, canine, bovine,feline, equine, primate, ovine, porcine and the like), with particularlypreferred mammals being humans. Nucleic acid sequences encoding Dkk-1include, but are not limited to human Dkk-1 (GenBank Accession Nos.AH009834, XM_(—)005730, AF261158, AF261157, AF177394, AF127563 andNM_(—)012242), Mus musculus dickkopf homolog 1 (GenBank Accession No.NM_(—)010051), and Danio rerio dickkopf-1 (GenBank Accession Nos.AF116852 and AB023488). The genomic sequences with exon annotation areGenBank Accession Nos. AF261157 and AF261158. Also contemplated arehomologs of these sequences which have Dkk-1 activity in the Wntpathway. Dkk-1 amino acid sequences include, but are not limited tohuman dickkopf homolog 1 (GenBank Accession Nos. AAG15544, BAA34651,NP_(—)036374, MF02674, AAD21087, and XP_(—)005730), Danio rerio(zebrafish) dickkopf1 (GenBank Accession Nos. BAA82135 and AAD22461) andmurine dickkopf-1 (GenBank Accession Nos. O54908 and NP_(—)034181).Variants and homologs of these sequences which possess Dkk-1 activityare also included when referring to Dkk-1.

[0239] By “Dkk mediated” disorder, condition or disease is any abnormalstate that involves Dkk activity. The abnormal state can be induced byenvironmental exposure or drug administration. Alternatively, thedisease or disorder can be due to a genetic defect. Dkk mediateddiseases, disorders and conditions include but are not limited to bonemass disorders or conditions and lipid disorders and conditions. Forexample, bone mass disorders/conditions/diseases, which may be mediatedby Dkk, include but are not limited to age related loss of bone, bonefractures (e.g., hip fracture, Colle's fracture, vertebral crushfractures), chondrodystrophies, drug-induced disorders (e.g.,osteoporosis due to administration of glucocorticoids or heparin andosteomalacia due to administration of aluminum hydroxide,anticonvulsants, or glutethimide), high bone turnover, hypercalcemia,hyperostosis, osteogenesis imperfecta, osteomalacia, osteomyelitis,osteoporosis, Paget's disease, osteoarthritis, and rickets.

[0240] Lipid disorders/diseases/conditions, which may be mediated byDkk, include but are not limited to familial lipoprotein lipasedeficiency, familial apoprotein CII deficiency, familial type 3hyperlipoproteinemia, familial hypercholesterolemia, familialhypertriglyceridemia, multiple lipoprotein-type hyperlipidemia, elevatedlipid levels due to dialysis and/or diabetes, and elevated lipid levelsof unknown etiologies

[0241] The term “recognizes and binds,” when used to define interactionsof antisense nucleotides, siRNAs (small inhibitory RNA), or shRNA (shorthairpin RNA) with a target sequence, means that a particular antisense,siRNA, or shRNA sequence is substantially complementary to the targetsequence, and thus will specifically bind to a portion of an mRNAencoding polypeptide. As such, typically the sequences will be highlycomplementary to the mRNA target sequence, and will have no more than 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 base mismatches throughout the sequence.In many instances, it may be desirable for the sequences to be exactmatches, i.e. be completely complementary to the sequence to which theoligonucleotide specifically binds, and therefore have zero mismatchesalong the complementary stretch. As such, highly complementary sequenceswill typically bind quite specifically to the target sequence region ofthe mRNA and will therefore be highly efficient in reducing, and/or eveninhibiting the translation of the target mRNA sequence into polypeptideproduct.

[0242] Substantially complementary oligonucleotide sequences will begreater than about 80 percent complementary (or ‘% exact-match’) to thecorresponding mRNA target sequence to which the oligonucleotidespecifically binds, and will, more preferably be greater than about 85percent complementary to the corresponding mRNA target sequence to whichthe oligonucleotide specifically binds. In certain aspects, as describedabove, it will be desirable to have even more substantiallycomplementary oligonucleotide sequences for use in the practice of theinvention, and in such instances, the oligonucleotide sequences will begreater than about 90 percent complementary to the corresponding mRNAtarget sequence to which the oligonucleotide specifically binds, and mayin certain embodiments be greater than about 95 percent complementary tothe corresponding mRNA target sequence to which the oligonucleotidespecifically binds, and even up to and including 96%, 97%, 98%, 99%, andeven 100% exact match complementary to the target mRNA to which thedesigned oligonucleotide specifically binds.

[0243] Percent similarity or percent complementary of any of thedisclosed sequences may be determined, for example, by comparingsequence information using the GAP computer program, version 6.0,available from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (1970). Briefly, the GAP program defines similarity as the numberof aligned symbols (i.e., nucleotides or amino acids) which are similar,divided by the total number of symbols in the shorter of the twosequences. The preferred default parameters for the GAP program include:(1) a unary comparison matrix (containing a value of 1 for identitiesand 0 for non-identities) for nucleotides, and the weighted comparisonmatrix of Gribskov and Burgess (1986), (2) a penalty of 3.0 for each gapand an additional 0.10 penalty for each symbol in each gap; and (3) nopenalty for end gaps.

[0244] By “mimetic” is meant a compound or molecule that performs thesame function or behaves similarly to the compound mimicked.

[0245] By “reporter element” is meant a polynucleotide that encodes apoplypeptide capable of being detected in a screening assays. Examplesof polypeptides encoded by reporter elements include, but are notlimited to, lacZ, GFP, luciferase, and chloramphenicolacetyltransferase.

[0246] 2. Introduction

[0247] A polymorphism in LRP5 (Zmax), G171V, designated as HBM, has beenidentified as conferring a high bone mass phenotype in a population ofrelated subjects as described in co-pending applications InternationalPatent Application PCT/US 00/16951, and U.S. patent application Ser.Nos. 09/543,771 and 09/544,398, which are hereby incorporated byreference in their entirety (Little et al., Am J Hum Genet. 70:11-19(2002)). LRP5 is also described in International Patent Application WO98/46743, which is incorporated by reference in its entirety. Loss ofLRP5 function has been shown to have a deleterious effect on bone (Gonget al., Cell 107:513-523 (2001)). Additionally, the HBM polymorphism andLRP5 may also be important in cardiac health and lipid-mediateddisorders. Thus, methods of regulating their activity can serve asmethods of treating and/or preventing cardiac and lipid-mediateddisorders.

[0248] Recent studies have indicated that LRP5 participates in the Wntsignal transduction pathway. The Wnt pathway is critical in limb earlyembryological development. A recently published sketch of the componentsof Wnt signaling is shown in FIG. 1 (Nusse, 2001http://www.stanford.edu/˜rnusse/pathways/cell2.html) (see also, Nusse,Nature 411:255-6 (2001); and Mao et al., Nature 411:321-5 (2001)).Briefly summarized, Wnt proteins are secreted proteins which interactwith the transmembrane protein Frizzled (Fz). LRP proteins, such as LRP5and LRP6, are believed to modulate the Wnt signal in a complex with Fz(Tamai et al., Nature 407:530-5 (2000)). The Wnt pathway actsintracellularly through the Disheveled protein (Dsh) which in turninhibits glycogen synthetase kinase-3 (GSK3) from phosphorylatingβ-catenin. Phosphorylated β-catenin is rapidly degraded followingubiquitination. However, the stabilized β-catenin accumulates andtranslocates to the nucleus where it acts as a cofactor of the T-cellfactor (TCF) transcription activator complex.

[0249] The protein dickkopf-1 (Dkk-1) is reported to be an antagonist ofWnt pathway. Dkk-1 is required for head formation in early development.Dkk-1 and its function in the Wnt pathway are described in e.g.,Krupnik, et al., Gene 238:301-13 (1999); Fedi et al., J. Biol. Chem.274:19465-72 (1999); see also for Dkk-1 and the Wnt pathway, Wu et al.,Curr. Biol. 10:1611-4 (2000), Shinya et al., Mech. Dev. 98:3-17 (2000),Mukhopadhyay et al., Dev Cell 1:423-434 (2001) and in PCT PatentApplication No. WO 00/52047, and in references cited in each. It hasbeen known that Dkk-1 acts upstream of Dsh, however the nature of themechanism of inhibition by Dkk-1 is just beginning to be elucidated.Dkk-1 is expressed in the mouse embryonic limb bud and its disruptionresults in abnormal limb morphogensis, among other developmental defects(Gotewold et al., Mech. Dev. 89:151-3 (1999); and, Mukhopadhyay et al.,Dev Cell 1:423-434 (2001)).

[0250] Related U.S. provisional application 60/291,311 disclosed a novelinteraction between Dkk-1 (GenBank Accession No. XM 005730) and LRP5.The interaction between Dkk-1 and LRP5 was discovered by a yeast twohybrid (Y2H) screen for proteins which interact with the ligand bindingdomain of LRP5, as described in Example 1. The two-hybrid screen is acommon procedure in the art, which is described, for example, by Gietzet al., Mol. Cell. Biochem. 172:67-79 (1997); Young, Biol. Reprod.58:302-11 (1998); Brent and Finley, Ann. Rev. Genet. 31:663-704 (1997);and Lu and Hannon, eds., Yeast Hybrid Technologies, Eaton Publishing,Natick Mass., (2000). More recently, other studies confirm that Dkk-1 isa binding partner for LRP and modulates the Wnt pathway via directbinding with LRP (R. Nusse, Nature 411:255-256 (2001); A. Bafico et al.,Nat. Cell Biol. 3:683-686 (2001); M. Semënov, Curr. Biol. 11:951-961(2001); B. Mao, Nature 411:321-325 (2001), Zorn, Curr. Biol. 11:R592-5(2001)); and, L. Li et al., J. Biol Chem. 277:5977-81 (2002)).

[0251] Mao and colleagues (2001) identified Dkk-1 as a ligand for LRP6.Mao et al. suggest that Dkk-1 and LRP6 interact antagonistically whereDkk proteins inhibit the Wnt coreceptor functions of LRP6. Usingco-immunoprecipitation, the group verified that the Dkk-1/LRP6interaction was direct. Dkk-2 was also found to directly bind LRP6.Contrary to data contained in provisional application 60/291,311, Mao etal. report that no interaction was detected between any Dkk protein andLRP5, as well as no interaction with LDLR, VLDLR, ApoER, or LRP).Additionally, Mao et al. demonstrated that LRP6 can titrate Dkk-1'seffects of inhibiting Wnt signaling using the commercial TCF-luciferasereporter gene assay (TOPFLASH). A similar conclusion was drawn fromanalogous studies in Xenopus embryos. Deletion analyses of LRP6functional domains revealed that EGF repeats (beta-propellers) 3 and 4were necessary for Dkk-1 binding and that the ligand binding domains ofLRP6 had no effect on Dkk-1 binding. The findings of Mao et al. contrastwith data obtained by the present inventors indication that the ligandbinding domains of LRP5 were necessary and sufficient for Dkk-1 bindingin yeast. Using classical biochemical ligand-receptor studies, Mao etal. determined a Kd=0.34 nM for Dkk-1/LRP6 and a Kd=0.73 nM forDkk-2/LRP6.

[0252] Semenov et al. (2001) verified the Mao group's results andconfirmed by coimmunoprecipitation that Dkk-1 does not directly bind toWnt or Frizzled but rather interacts with LRP6. Their Scatchard analysesfound a Kd=0.5 nM for Dkk-1/LRP6. Semenov et a/. also demonstrated thatDkk-1 could abolish an LRP5/Frizzled8 complex implying that Dkk-1 canalso repress Wnt signaling via interactions with LRP5. A Dkk-1 mutantwhere cysteine 220 was changed to alanine abolished LRP6 binding and wasunable to repress Wnt signaling. Studies in Xenopus embryos confirmedthe results and revealed a functional consequence of Dkk-1/LRP6:repression of Wnt signaling. Their Xenopus work also suggested thatLRP6/Dkk-1 may be specific for the canonical, -catenin-mediated, Wntpathways as opposed to the Wnt Planar Cell Polarity pathway.

[0253] Bafico et al. (2001) employed a ¹²⁵I-labeled Dkk-1 molecule toidentify LRP6 as its sole membrane receptor with a Kd=0.39 nM. Again,the functional consequences of the Dkk-1/LRP6 interaction was arepression of the canonical Wnt signaling even when Dkk-1 was added atextremely low concentrations (30 pM).

[0254] Not wishing to be bound by theory, it is believed that thepresent invention provides an explanation for the mechanism of Dkk-1inhibition of the Wnt pathway and provides a mechanism whereby the Wntpathway may be modulated. The present application and relatedprovisional application 60/291,311 describe Dkk-1/LRP5 interactions anddemonstrate that the interaction between LRP5/LRP6/HBM and Dkk can beused in a method as an intervention point in the Wnt pathway for ananabolic bone therapeutic or a modulator of lipid metabolism.

[0255] As detailed below, in the section “Methods to Identify BindingPartners” and Examples 6 and 7, Dkk-1 is able to repress LRP5-mediatedWnt signaling but not HBM-mediated Wnt signaling. This observation is ofparticular interest because the HBM mutation in LRP5 is a gain offunction or activation mutation. That is, Wnt signaling, via thecanonical pathway, is enhanced with HBM versus LRP5. The present datasuggest the mechanism of this functional activation: the inability ofDkk-1 to repress HBM-mediated Wnt signaling. Further investigations ofother Wnt or Dkk family members show differential activities in thecanonical Wnt pathway that demonstrate the complexity and variability inWnt signaling that can be achieved depending on the LRP/Dkk/Wnt/Frizzledrepertoire that is expressed in a particular cell or tissue. This mayattest to the apparent bone specificity of the HBM phenotype in humansand in the HBM transgenic animals.

[0256] Furthermore, the present data reveal the importance andfunctional consequence for the potential structural perturbation of thefirst beta-propeller domain of LRP5. Our data identified the ligandbinding domain of LRP5 as the interacting region with Dkk-1 while theMao et al. publication demonstrated the functional role of propellers 3and 4 in their LRP6/Dkk-1 studies. In the present invention, weimplicate the first beta propeller domain, via the HBM mutation atresidue 171, as having a functional consequence in the Dkk-1 -mediatedWnt pathway. The involvement of position 171 of propeller 1 may bedirect or indirect with Dkk-1. Direct involvement could arise fromperturbations of the 3-dimensional structure of the HBM extracellulardomain that render Dkk-1 unable to bind. Alternatively, residue 171 ofpropeller 1 may directly interact with Dkk-1; however, by itself, it isinsufficient to bind and requires other LRP5 domains. Potential indirectcandidate molecules may be among the proteins identified the Dkk-1yeast-two-hybrid experiments.

[0257] It may be that the disruption of Dkk activity is not necessarilymediated by enhancing or preventing the binding of Dkk to LRP5/LRP6/HBM.More than one mechanism may be involved. Indeed, the inventors haveobserved that Dkk-1 binds LRP5, LRP6, and HBM. It is able to effectivelyinhibit LRP6, and to a slightly lesser extent, LRP5 activity. Further,has been observed that different members of the Dkk familydifferentially affect LRP5/LRP6/HBM activity. For example, Dkk-1inhibits LRP5/LRP6/HBM activity while another Dkk may enhanceLRP5/LRP6/HBM activity. An endpoint to consider is the modulation of theLRP5/LRP6/HBM activity, not simply binding.

[0258] The present disclosure shows that targeting the disruption of theDkk-1/LRP5 interaction is a therapeutic intervention point for an HBMmimetic agent. A therapeutic agent of the invention may be a smallmolecule, peptide or nucleic acid aptamer, antibody, or otherpeptide/protein, etc. Methods of reducing Dkk-1 expression may also betherapeutic using methodologies such as: RNA interference, antisenseoligonucleotides, morpholino oligonucleotides, PNAs, antibodies to Dkk-1or Dkk-1 interacting proteins, decoy or scavenger LRP5 or LRP6receptors, and knockdown of Dkk-1 or Dkk-1 interactor transcription.

[0259] In an embodiment of the present invention, the activity of Dkk-1or the activity of a Dkk-1 interacting protein may be modulated forexample by binding with a peptide aptamer of the present invention. Inanother embodiment, LRP5 activity may be modulated by a reagent providedby the present invention (e.g., a peptide aptamer). In anotherembodiment, the Dkk-1/LRP5 interaction may be modulated by a reagent ofthe present invention (e.g., a Dkk-1 interacting protein such as thoseidentified in FIG. 5). In another embodiment, the Wnt signaltransduction pathway may be modulated by use of one or more of the abovemethods. In a preferred embodiment of the present invention, the Dkk-1mediated activity of the Wnt pathway may be specifically modulated byone or more of the above methods. In another preferred embodiment of thepresent invention, the Wnt signal transduction pathway may be stimulatedby down-regulating Dkk-1 interacting protein activity; suchdown-regulation could, for example, yield greater LRP5 activity. In amore preferred embodiment, by stimulating LRP5 activity, bone massregulation may be stimulated to restore or maintain a more optimallevel. In another preferred embodiment, by stimulating LRP5 activity,lipid metabolism may be stimulated to restore or maintain a more optimallevel. Alternative embodiments provide methods for screening candidatedrugs and therapies directed to correction of bone mass disorders orlipid metabolism disorders. And, preferred embodiments of the presentinvention provide drugs and therapies developed by the use of thereagents and/or methods of the present invention. One skilled in the artwill understand that the present invention provides important researchtools to develop an effective model of osteoporosis, to increaseunderstanding of bone mass and lipid modulation, and to modulate bonemass and lipid metabolism.

[0260] Previous investigation of a large family in which high bone massis inherited as a single gene (autosomal dominant) trait (HBM-1) hasprovided important insight into the mechanism by which bone densitymight be modulated. Members of this family have significantly increasedspinal and hip BMD (>3 standard deviations above the norm) which affectsyoung adults as well as elderly family members into the ninth decade.The bones of affected members, while appearing very denseradiographically, have normal external shape and outer dimensions.Cortical bone is thickened on endosteal surfaces and “affected”individuals are asymptomatic without any other phenotypic abnormalities.Assays of biochemical markers that reflect skeletal turnover suggestthat the disorder is associated with a normal rate of bone remodeling.Affected individuals have achieved a balance in bone turnover at adensity that is significantly greater than necessary for normal skeletalstresses. Importantly, the bones most affected are load-bearing boneswhich are subjected to the greatest mechanical and gravitationalstresses (spine and hip). These are the most important bones to targetfir therapeutic interventions in osteoporosis. The gene identified asbeing responsible for this phenotype, Zmax or LRP5, was not previouslyassociated with bone physiology. The fact that modification of thisgene, such as that produced by the polymorphism leading to the autosomaldominant inheritance of the HBM family phenotype, identifies Zmax/LRP5and the pathway by which it is regulated, including DkkNVnt pathwaysdiscussed above, as an important target for developing modulators ofbone density. Modulation of Zmax/LRP5 to mimic the gain in functionprovided by the HBM polymorphism would be expected to provide animportant therapy for bone wasting conditions. Additionally, suchmodulation in young adults could enhance peak bone mass and prevent ordelay fracture risk later in life. Alternatively, modulation to reducefunction could be employed to treat conditions where bone is beinginappropriately produced.

[0261] 3. Polypeptides

[0262] Polypeptides contemplated for use in this invention include thosewhich modulate Dkk and Dkk interacting protein activities. Preferredpolypeptides and peptides include those which modulate the Wnt pathway.Examples of preferred sequences include the Y2H baits exemplified inFIG. 2, peptide aptamers of FIG. 3 (SEQ ID NOs:171-188) and FIG. 4 (SEQID NOs:189-192), the polypeptides of the Dkk-1 interacting proteinsidentified in FIG. 5, those polypeptides shown in FIG. 6, the LRPbinding domain of Dkk (amino acids 138-266 of hDkk1), the cysteine-richdomain 2 (a.a. 183-245 of hDkk-1), the cysteine-rich domain 1 (a.a.97-138 of hDkk), and LRP5 binding aptamers of FIG. 13 (including SEQ IDNOs:204-213). Although Dkk-1 is exemplified, the other Dkk proteinscontain substantially similar regions and may also be used according tothe present invention.

[0263] For example, the baits depicted in FIG. 2 were used in a yeasttwo hybrid (Y2H) screen. The Y2H screen was performed as described inExample 2 to determine the minimum required binding domain for Dkk-1 tobind LRP5. The minimum binding domain constructs (i.e., residues 139-266in bold below and residues 97-245 which are underlined, of Dkk-1)include the second cysteine rich domain which has sequence homology to acolipase fold. (SEQ ID NO: 128) mmalgaagat rvfvamvaaa lgghpllgvsatlnsvlnsn aiknlppplg gaaghpgsav 60 → saapgilypg gnkyqtidny qpypcaedeecgtdeycasp trggdagvgqi clacrkrrkr 120 cmrhamccpg nyckngic

_

_

_

_

180

_

_

_

_

_

240

 

 

 (GenBank Accession No. XP_005730).

[0264] This homology suggests a lipid-binding function and mayfacilitate Dkk-1 interactions at the plasma membrane (van Tilbeurgh, H.,Biochim. Biophys. Acta. 1441:173-84 (1999)). An interaction domain ofDkk-1 that is able to interact with the ligand binding domain (LBD) ofLRP5 is a useful reagent in the modulation of LRP5 activity andmodulation of Dkk-1/LRP5 complex formation. Similar screens can beprepared for Dkk-1 and Dkk-1 interacting proteins or polypeptides.

[0265] A set of peptide aptamers was identified from a library of randompeptides constrained and presented in a thioredoxin A (trxA) scaffold asdescribed in Example 3. Peptide aptamers are powerful new tools formolecular medicine as reviewed by Hoppe-Seyler & Butz, J. Mol. Med.,78:426-430 (2000); Brody and Gold, Rev. Mol. Biotech., 74:5-13 (2000);and Colas, Curr. Opin. in Chem. Biol. 4:54-9 (2000) and the referencescited therein. Briefly, peptide aptamers have been shown to be highlyspecific reagents capable of binding in vivo. As such, peptide aptamersprovide a method of modulating the function of a protein and may serveas a substitute for conventional knock-out methods, knock-down orcomplete loss of function. Peptide aptamers are also useful reagents forthe validation of targets for drug development and may be used astherapeutic compounds directly or provide the necessary foundation fordrug design. Once identified, the peptide insert may be synthesized andused directly or incorporated into another carrier molecule. Referencesreviewed and cited by Brody and Gold (2000, supra) describe demonstratedtherapeutic and diagnostic applications of peptide aptamers and would beknown to the skilled artisan.

[0266] The peptide aptamers of the present invention are useful reagentsin the binding of Dkk-1 to its ligands and thereby modulation of the Wntpathway and may be used to prevent Dkk-1 from inhibiting LRP5 modulationor Dkk-1 interacting protein modulation of the Wnt pathway. The sequenceof these peptide aptamers is shown in FIG. 3 (SEQ ID NOs:171-188). Thepeptide aptamers refers to the peptide constrained by the thioredoxinscaffold. The aptamers are also contemplated as therapeutic agents totreat Dkk-1 mediated diseases and conditions. Such aptamers are usefulstructural guides to chemists, for the design of mimetic compounds ofthe aptamers.

[0267] Peptide aptamers were likewise developed to the LRP5 ligandbinding domain (LBD) bait sequences. The sequences of these peptideaptamers is shown in FIG. 4 (SEQ ID NOs:189-192). These are usefulreagents which may be used to disrupt the Dkk-1/LRP5 binding interfacewhile leaving Dkk-1 undisturbed. These can be used as comparativecontrols for Wnt signaling, thus, a control is provided for thespecificity of any drug or therapy screened. The aptamers are alsouseful therapeutic agents to treat LRP mediated diseases and conditions.Such aptamers may also be used as structural guides to chemists, for thedesign of mimetic compounds of the aptamers.

[0268] Thirty proteins were identified which interact with Dkk-1, Dkk-1interacting proteins, were identified in a yeast-two-hybrid screen usingthe Dkk-1 bait and are shown in FIG. 5. It was noted that these resultssuggest an interaction of Dkk-1 with Notch-2. It has been suggested thatcross-talk exists between the Wnt and Notch signaling pathways. Forinstance, Presenilin1 (Ps1) is required for Notch processing andinhibits the downstream Wnt pathway. The extracellular domain of Notchis thought to interact with Wnt. Furthermore, the Notch intracellulardomain is thought to interact with disheveled and in signal inducedprocessing, the intracellular domain is thought to interact withpresenilin. (Soriano et a., J. Cell Biol. 152:785-94 (2001)). Foradditional information regarding the relationships between Notch and Wntsignaling, see Wesley, Mol. Cell. Biol. 19:5743-58 (1999) and Axelrod etal., Science 271:1826-32 (1996).

[0269] An interaction between Dkk-1 and chordin has also been noted;suggesting that cross-talk exists between the Wnt and TGF-beta/BMPsignaling pathways (Letamendia et al., J. Bone Joint Surg. Am. 83A:S31(2001); Labbe et al., Proc. Natl. Acad. Sci. USA 97:8358-63 (2000);Nishita et al., Nature 403:781-5 (2000); DeRobertis et al., Int. J. Dev.Biol.. 45:1389-97 (2001); and Saint-Jeannet et a., Proc. Natl. Acad.Sci. USA 94:13713-8 (1997)). The BMP signaling pathway has anestablished role in bone and connective tissue development, repair andhomeostasis (review in Rosen and Wozney “Bone Morphogenetic Proteins”In: Principles of Bone Biology, 2^(nd) Edition, Eds. J. Bilezikian, L.Raisz and G. Rodan, Academic Press, pp. 919-28 (2002)). Chordin is animportant molecule during development which also modulates BMP signalingin adults by sequestering BMPs in latent complexes (Piccolo et al., Cell86:589-98 (1996) reviewed in Reddi, Arthritis Res. 3:1-5 (2001);DeRobertis et al., Int. J. Dev. Biol. 45:189-97 (2001)). It may be thatDkk effects bone mass modulation through both the Wnt signaling pathwayvia LRP and the BMP pathway via chordin.

[0270] Moreover, a number of putative growth factors, growth factorrelated proteins, and extracellular matrix proteins have been identifiedas Dkk-1 interacting proteins. Additional information regarding Dkk-1interacting proteins identified in the Y2H assay may be obtained frompublicly available databases such as PubMed via the use of the accessionnumbers provided in the present application. In a preferred embodimentof the invention, the amino acid sequences of these Dkk-1 interactingproteins or biologically active fragments thereof be used to modulateDkk, Dkk-1, LRP5, LRP6, HBM, or Wnt activity. Although these proteinswere identified as interacting with Dkk-1, due to the substantialhomology between the various Dkk proteins, such interacting proteins arecontemplated to interact with the other Dkk family members.

[0271] 4. Aptamer Mimetics

[0272] The present invention further provides for mimetics of Dkk,particularly Dkk-1, and LRP5 peptide aptamers. Such aptamers may serveas structural guides to chemists for the design of mimetic compounds ofthe aptamers. The aptamers and their mimetics are useful as therapeuticagents to treat LRP- or Dkk-mediated diseases and conditions.

[0273] 5. Nucleic Acid Molecules

[0274] The present invention further provides nucleic acid moleculesthat encode polypeptides and proteins which interact with Dkk and Dkkinteracting proteins, and/or LRP5 (also LRP6 and HBM) to modulatebiological activities of these proteins. Preferred embodiments providenucleic acids encoding for fragments of Dkk-1 protein, including thenucleic acids of FIG. 7, the Dkk-1 interacting proteins listed in FIG.5, polypeptide aptamers of Dkk-1 (FIG. 3—SEQ ID NOs:171-188), LRP5 (FIG.4—SEQ ID NOs:189-192), FIG. 13 peptide aptamers (including SEQ IDNO:204-214) encoded by FIG. 12 polynucleotides (including SEQ IDNO:193-203), LRP6 and HBM and the related fusion proteins hereindescribed, preferably in isolated or purified form. As used herein,“nucleic acid” is defined as RNA, DNA, or cDNA that encodes a peptide asdefined above, or is complementary to a nucleic acid sequence encodingsuch peptides, or hybridizes to either the sense or antisense strands ofthe nucleic acid and remains stably bound to it under appropriatestringency conditions. The nucleic acid may encode a polypeptide sharingat least about 75% sequence identity, preferably at least about 80%, andmore preferably at least about 85%, with the peptide sequences; at leastabout 90%, 95%, 96%, 97%, 98%, and 99% or greater are also contemplated.Specifically contemplated are genomic DNA, cDNA, mRNA, antisensemolecules, enzymatically active nucleic acids (e.g., ribozymes), as wellas nucleic acids based on an alternative backbone or includingalternative bases, whether derived from natural sources or synthesized.Such hybridizing or complementary nucleic acids, however, are definedfurther as being novel and nonobvious over any prior art nucleic acidincluding that which encodes, hybridizes under appropriate stringencyconditions, or is complementary to a nucleic acid encoding a proteinaccording to the present invention.

[0275] As used herein, the terms “hybridization” (hybridizing) and“specificity” (specific for) in the context of nucleotide sequences areused interchangeably. The ability of two nucleotide sequences tohybridize to each other is based upon the degree of complementarity ofthe two nucleotide sequences, which in turn is based on the fraction ofmatched complementary nucleotide pairs. The more nucleotides in a givensequence that are complementary to another sequence, the greater thedegree of hybridization of one to the other. The degree of hybridizationalso depends on the conditions of stringency which include temperature,solvent ratios, salt concentrations, and the like. In particular,“selective hybridization” pertains to conditions in which the degree ofhybridization of a polynucleotide of the invention to its target wouldrequire complete or nearly complete complementarity. The complementaritymust be sufficiently high so as to assure that the polynucleotide of theinvention will bind specifically to the target nucleotide sequencerelative to the binding of other nucleic acids present in thehybridization medium. With selective hybridization, complementarity willbe about 90-100%, preferably about 95-100%, more preferably about 100%.

[0276] “Stringent conditions” are those that (1) employ low ionicstrength and high temperature for washing, for example: 0.015 M NaCl,0.0015 M sodium titrate, 0.1% SDS at 50° C.; or (2) employ duringhybridization a denaturing agent such as formamide, for example, 50%(vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750mM NaCl, 75 mM sodium citrate at 42° C. Another example is use of 50%formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodiumphosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution,sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfateat 42° C., with washes at 42° C. in 0.2× SSC and 0.1% SDS. A skilledartisan can readily determine and vary the stringency conditionsappropriately to obtain a clear and detectable hybridization signal.

[0277] As used herein, a nucleic acid molecule is said to be “isolated”or “purified” when the nucleic acid molecule is substantially separatedfrom contaminant nucleic acid encoding other polypeptides from thesource of nucleic acid. Isolated or purified is also meant to includenucleic acids which encode Dkk or fragments thereof which lacksurrounding genomic sequences that flank the Dkk gene. Isolated orpurified is further intended to include nucleic acids which encode Dkkinteracting proteins or biologically active fragments thereof which lacksurrounding genomic sequences that flank the Dkk interacting proteingenes.

[0278] The present invention further provides fragments of the encodingnucleic acid molecule. As used herein, a fragment of an encoding nucleicacid molecule refers to a small portion of the entire protein encodingsequence. The size of the fragment will be determined by the intendeduse. For example, if the fragment is chosen so as to encode an activeportion of the protein, the fragment will need to be large enough toencode the functional region(s) of the protein. If the fragment is to beused as a nucleic acid probe or PCR primer, then the fragment length ischosen so as to obtain a relatively small number of false positivesduring probing/priming.

[0279] Fragments of the encoding nucleic acid molecules of the presentinvention (i.e., synthetic oligonucleotides) that are used as probes orspecific primers for the polymerase chain reaction (PCR), or tosynthesize gene sequences encoding proteins of the invention can easilybe synthesized by chemical techniques, for example, the phosphotriestermethod of Matteucci et al. (J. Am. Chem. Soc. 103:3185-3191 (1981)) orusing automated synthesis methods. In addition, larger DNA segments canreadily be prepared by well known methods, such as synthesis of a groupof oligonucleotides that define various modular segments of the gene,followed by ligation of oligonucleotides to build the complete modifiedgene.

[0280] The polypeptide encoding nucleic acid molecules of the presentinvention may further be modified to contain a detectable label fordiagnostic and probe purposes. A variety of such labels are known in theart and can readily be employed with the encoding molecules hereindescribed. Suitable labels include, but are not limited to, biotin,radiolabeled nucleotides and the like. A skilled artisan can employ anyof the art known labels to obtain a labeled encoding nucleic acidmolecule.

[0281] Modifications to the primary structure itself by deletion,addition, or alteration of the amino acids incorporated into the proteinsequence during translation can be made without destroying the activityof the protein. Such substitutions or other alterations result inproteins having an amino acid sequence encoded by a nucleic acid fallingwithin the contemplated scope of the present invention.

[0282] Antisense molecules corresponding to the polypeptide coding orcomplementary sequence may be prepared. Methods of making antisensemolecules which bind to mRNA, form triple helices or are enzymaticallyactive and cleave TSG RNA and single stranded DNA (ssDNA) are known inthe art. See, e.g., Antisense and Ribozyme Methodology:LaboratoryCompanion (Ian Gibson, ed., Chapman & Hall, 1997) and RibozymeProtocols: Methods in Molecular Biology (Phillip C. Turner, ed., HumanaPress, Clifton, N.J., 1997).

[0283] Also contemplated is the use of compounds which mediatepostranscriptional gene silencing (PTGS), quelling and RNA interference(RNAi). These compounds typically are about 21 to about 25 nucleotidesand are also known as short interfering RNAs or short inhibitory RNAs(siRNAs). The siRNAs are produced from an initiating double stranded RNA(dsRNA). Although the full mechanism by which the siRNAs function is notfully elucidated, it is known that these siRNAs transform the targetmRNA into dsRNA, which is then degraded. Preferred forms are 5′phosphorylated siRNAs, however, hydroxylated forms may also be utilized.For additional background regarding the preparation and mechanism ofsiRNAs generally, see, e.g., Lipardi et al., Cell 107(3): 297-307(2001); Boutla et al., Curr. Biol. 11 (22): 1776-80 (2001); Djikeng etal., RNA 7(11): 1522-30 (2001); Elbashir et al., EMBO J. 20(23): 6877-88(2001); Harborth et al., J. Cell. Sci. 114(Pt. 24): 4557-65 (2001);Hutvagner et al., Science 293(5531): 811-3 (2001); and Elbashir et al.,Nature 411:494-98 (2001).

[0284] Also contemplated are short hairpin RNAs (shRNAs). shRNAs are amodification of the siRNA method described above. Instead oftransfecting exogenously synthesized dsRNA into a cell,sequence-specific silencing can be achieved by stabling expressing siRNAfrom a DNA template as a fold-back stem-loop, or hairpin. This approachis known as shRNA. This method permits the analysis of loss of functionphenotypes due to sequence-specific gene silencing in mammalian cells byavoiding many of the problems associated with siRNAs, such as RNasedegradation of the reagents, expensive chemical synthesis, etc. Foradditional background regarding the preparation and mechanism of shRNAsgenerally, see, e.g., Yu et al., PNAS 99:6047-6052 (2002); Paddison etal., Genes and Devel. 16:948-58 (2002); and Brummelkamp et al., Science296:550-553 (2002). For additional background on the use of this methodin mammalian gene knockdown methodologies, see Tuschl, Nature Biotech.20:446-448 (2002) (and references therein).

[0285] In one preferred embodiment, the siRNA or shRNA is directed to aDkk encoding mRNA, wherein a preferred Dkk is Dkk-1. In anotherembodiment, the siRNA or shRNA is directed towards a protein which bindsto and modulates the activity of or is modulated by a Dkk; theseproteins include LRP5, LRP6 and HBM as well as other members of the Wntpathway.

[0286] 6. Isolation of Other Related Nucleic Acid Molecules

[0287] The identification of the nucleic acid molecule of Dkk allows askilled artisan to isolate nucleic acid molecules that encode othermembers of the Dkk family (see, Krupnik et al., 1999). Further, thepresently disclosed nucleic acid molecules allow a skilled artisan toisolate nucleic acid molecules that encode Dkk-1 -like proteins, inaddition to Dkk-1. The presently disclosed Dkk-1 interacting proteinsand their corresponding nucleic acid molecules allows a skilled artisanto further isolate other related protein family members which interactwith Dkk-1.

[0288] A skilled artisan can readily use the amino acid sequence of Dkkand Dkk interacting proteins to generate antibody probes to screenexpression libraries prepared from appropriate cells. Typically,polyclonal antiserum from mammals such as rabbits immunized with thepurified protein (as described below) or monoclonal antibodies can beused to probe a mammalian cDNA or genomic expression library, such as ahuman macrophage library, to obtain the appropriate coding sequence forother members of the protein family. The cloned cDNA sequence can beexpressed as a fusion protein, expressed directly using its own controlsequences, or expressed by constructions using control sequencesappropriate to the particular host used for expression of the desiredprotein.

[0289] Alternatively, a portion of the coding sequence herein describedcan be synthesized and used as a probe to retrieve DNA encoding a memberof the protein family from any mammalian organism. Oligomers containingapproximately 18-20 nucleotides (encoding about a 6-7 amino acidstretch) are prepared and used to screen genomic DNA or cDNA librariesto obtain hybridization under stringent conditions or conditions ofsufficient stringency to eliminate an undue level of false positives.

[0290] Additionally, pairs of oligonucleotide primers can be preparedfor use in a polymerase chain reaction (PCR) to selectively clone anencoding nucleic acid molecule. A PCR denature/anneal/extend cycle forusing such PCR primers is well known in the art and can readily beadapted for use in isolating other encoding nucleic acid molecules. Forexample, degenerate primers can be utilized to obtain sequences relatedto Dkk-1 or Dkk-1 interacting proteins. Primers can be designed that arenot perfectly complementary and can still hybridize to a portion of atarget sequence or flanking sequence and thereby provide foramplification of all or a portion of a target sequence. Primers of about20 nucleotides or less, preferably have about one to three mismatcheslocated at the 5′ and/or 3′ ends. Primers of about 20 to 30 nucleotideshave up to about 30% mismatches and can still hybridize to a targetsequence. Hybridization conditions for primers with mismatch can bedetermined by the method described in Maniatis et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1982) or by reference to known methods. The ability of theprimer to hybridize to a sequence of either Dkk-1, a Dkk-1 interactingprotein, or a related sequence under varying conditions can bedetermined using this method. Because a target sequence is known, theeffect of mismatches can be determined by methods known to those ofskill in the art. Degenerate primers would be based on putativeconserved amino acid sequences of the Dkk-1 and Dkk-1 interactingprotein genes.

[0291] 7. rDNA Molecules for Polypeptide Expression

[0292] The present invention further provides recombinant DNA molecules(rDNAs) that contain a polypeptide coding sequence. As used herein, arDNA molecule is a DNA molecule that has been subjected to molecularmanipulation in situ. Methods for generating rDNA molecules are wellknown in the art, for example, see Sambrook et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989). In the preferred rDNA molecules, a coding DNA sequence isoperably linked to expression control sequences and/or vector sequences.

[0293] The choice of vector and/or expression control sequences to whichone of the protein family encoding sequences of the present invention isoperably linked depends directly, as is well known in the art, on thefunctional properties desired, e.g., protein expression, and the hostcell to be transformed. A vector contemplated by the present inventionis at least capable of directing the replication and/or insertion intothe host chromosome, and preferably also expression, of the structuralgene included in the rDNA molecule.

[0294] Expression control elements that are used for regulating theexpression of an operably linked protein encoding sequence are known inthe art and include, but are not limited to, inducible promoters,constitutive promoters, secretion signals, and other regulatoryelements. Preferably, the inducible promoter is readily controlled, suchas being responsive to a nutrient in the host cell's medium. Preferredpromoters include yeast promoters, which include promoter regions formetallothionein, 3-phosphoglycerate kinase or other glycolytic enzymessuch as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymesresponsible for maltose and galactose utilization, and others. Vectorsand promoters suitable for use in yeast expression are further describedin EP 73,675A. Appropriate non-native mammalian promoters might includethe early and late promoters from SV40 (Fiers et al, Nature, 273:113(1978)) or promoters derived from Moloney murine leukemia virus, mousetumor virus, avian sarcoma viruses, adenovirus 11, bovine papillomavirus or polyoma. In addition, the construct may be joined to anamplifiable gene (e.g., DHFR) so that multiple copies of the gene may bemade. For appropriate enhancer and other expression control sequences,see also Enhancers and Eukaryotic Gene Expression (Cold Spring HarborPress, Cold Spring Harbor, N.Y., 1983). Preferred bone related promotersinclude CMVbActin or type I collagen promoters to drive expression ofthe human HBM, Zmax1/LRP5 or LRP6 cDNA. Other preferred promoters formammalian expression are from cytomegalovirus (CMV), Rous sarcoma virus(RSV), Simian virus 40 (SV40), and EF-1a (human elongation factor1a-subunit).

[0295] In one embodiment, the vector containing a coding nucleic acidmolecule will include a prokaryotic replicon, i.e., a DNA sequencehaving the ability to direct autonomous replication and maintenance ofthe recombinant DNA molecule extrachromosomally in a prokaryotic hostcell, such as a bacterial host cell, transformed therewith. Suchreplicons are well known in the art. In addition, vectors with aprokaryotic replicon may also include a gene whose expression confers adetectable marker such as a drug resistance. Typical bacterial drugresistance genes are those that confer resistance to ampicillin ortetracycline.

[0296] Vectors that include a prokaryotic replicon can further include aprokaryotic or bacteriophage promoter capable of directing theexpression (transcription and translation) of the coding gene sequencesin a bacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Typical of such vector plasmids are pUC8,pUC9, pBR322 and pBR329 available from Biorad Laboratories, (Richmond,Calif.), and pPL and pKK223 available from Pharmacia (Piscataway, N.J.).

[0297] Expression vectors compatible with eukaryotic cells, preferablythose compatible with vertebrate cells, can also be used to form a rDNAmolecule that contains a coding sequence. Eukaryotic cell expressionvectors are well known in the art and are available from severalcommercial sources. Typically, such vectors are provided containingconvenient restriction sites for insertion of a desired DNA segment.Typical of such vectors are pSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d(International Biotechnologies, Inc.), vector systems that includeHistidine Tags and periplasmic secretion, or other vectors described inthe art.

[0298] Eukaryotic cell expression vectors used to construct the rDNAmolecules of the present invention may further include a selectablemarker that is effective in an eukaryotic cell, preferably a drugresistance selection marker. A preferred drug resistance marker is thegene whose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene (Southern et al., J. Mol. Anal. Genet.1:327-341 (1982)). Alternatively, the selectable marker can be presenton a separate plasmid, and the two vectors introduced by co-transfectionof the host cell, and selected by culturing in the appropriate drug forthe selectable marker.

[0299] 8. Host Cells Containing an Exogenously Supplied rDNA NucleicAcid Molecule

[0300] The present invention further provides host cells transformedwith a nucleic acid molecule that encodes a polypeptide or protein ofthe present invention. The host cell can be either prokaryotic oreukaryotic. Eukaryotic cells useful for expression of a protein of theinvention are not limited, so long as the cell line is compatible withcell culture methods and compatible with the propagation of theexpression vector and expression of the gene product. Preferredeukaryotic host cells include, but are not limited to, yeast, insect andmammalian cells, preferably vertebrate cells such as those from a mouse,rat, monkey or human cell line but also can include invertebrates with,for example, cartilage. Preferred eukaryotic host cells include but arenot limited to Chinese hamster ovary (CHO) cells (ATCC No. CCL61), NIHSwiss mouse embryo cells NIH/3T3 (ATCC No. CRL 1658), baby hamsterkidney cells (BHK), HOB-03-CE6 osteoblast cells, and other likeeukaryotic tissue culture cell lines.

[0301] Any prokaryotic host can be used to express a rDNA moleculeencoding a protein of the invention. A preferred prokaryotic host is Ecoli.

[0302] Transformation of appropriate cell hosts with a recombinant DNA(rDNA) molecule of the present invention is accomplished by well knownmethods that typically depend on the type of vector used and host systememployed. With regard to transformation of prokaryotic host cells,electroporation and salt treatment methods are typically employed; see,for example, Cohen et al., Proc. Natl. Acad. Sc. USA 69: 2110 (1972);Maniatis et al. (1982); and Sambrook et al. (1989). With regard totransformation of vertebrate cells with vectors containing rDNAs,electroporation, cationic lipid or salt treatment methods are typicallyemployed; see, for example, Graham et al., Virol. 52: 456 (1973); Wigleret al., Proc. Natl. Acad. Sci. USA 76: 1373-76 (1979).

[0303] Successfully transformed cells, i.e., cells that contain a rDNAmolecule of the present invention, can be identified by well knowntechniques including the selection for a selectable marker. For example,cells resulting from the introduction of an rDNA of the presentinvention can be cloned to produce single colonies. Cells from thosecolonies can be harvested, lysed and their DNA content examined for thepresence of the rDNA using a method such as that described by Southern,J. Mol. Biol. 98: 503 (1975), or Berent et al., Biotech. 3: 208 (1985).Alternatively, the cells can be cultured to produce the proteins encodedby the rDNA and the proteins harvested and assayed, using for example,any suitable immunological method. See, e.g., Harlow et al., (1988).

[0304] Recombinant DNA can also be utilized to analyze the function ofcoding and non-coding sequences. Sequences that modulate the translationof the mRNA can be utilized in an affinity matrix system to purifyproteins obtained from cell lysates that associate with the Dkk-1 orDkk-1 interacting protein or expression control sequence. Syntheticoligonucleotides would be coupled to the beads and probed with thelysates, as is commonly known in the art. Associated proteins could thenbe separated using, for example, a two dimensional SDS-PAGE system.Proteins thus isolated could be further identified using massspectroscopy or protein sequencing. Additional methods would be apparentto the skilled artisan.

[0305] 9. Production of Recombinant Peptides and Proteins using a cDNAor Other Recombinant Nucleic Acids

[0306] The invention also relates to nucleic acid molecules which encodea Dkk protein and polypeptide fragments thereof, and proteins andpolypeptides which bind to Dkk -(e.g., LRP5, LRP6 and HBM, Dkkinteracting proteins such as the proteins of FIG. 5) and molecularanalogues. The polypeptides of the present invention include the fulllength Dkk and polypeptide fragments thereof, Dkk binding proteins andpolypeptides thereof. Preferably these proteins are mammalian proteins,and most preferably human proteins and biologically active fragmentsthereof. Alternative embodiments include nucleic acid molecules encodingpolypeptide fragments having a consecutive amino acid sequence of atleast about 3, 5, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90,100, 125, 150, 175, or 200 amino acid residues from a common polypeptidesequence; amino acid sequence variants of a common polypeptide sequencewherein an amino acid residue has been inserted N- or C-terminal to, orwithin, the polypeptide sequence or its fragments; and amino acidsequence variants of the common polypeptide sequence or its fragments,which have been substituted by another conserved residue. Recombinantnucleic acid molecules which encode polypeptides include thosecontaining predetermined mutations by, e.g., homologous recombination,site-directed or PCR mutagenesis, and recombinant Dkk proteins orpolypeptide fragments of other animal species, including but not limitedto vertebrates (e.g., rabbit, rat, murine, porcine, camelid, reptilian,caprine, avian, fish, bovine, ovine, equine and non-human primatespecies) as well as invertebrates, and alleles or other naturallyoccurring variants and homologs of Dkk binding proteins of the foregoingspecies and of human sequences. Also contemplated herein are derivativesof the commonly known Dkk, Dkk interacting proteins, or fragmentsthereof, wherein Dkk, Dkk interacting proteins, or their fragments havebeen covalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid (for example a detectable moiety such as an enzyme or radioisotope)and soluble forms of Dkk. It is further contemplated that the presentinvention also includes nucleic acids with silent mutations which willhybridize to the endogenous sequence and which will still encode thesame polypeptide.

[0307] The nucleic acid molecules encoding Dkk binding proteins, theLRP5 binding domain fragment of Dkk, or other polypeptides of thepresent invention are preferably those which share a common biologicalactivity (e.g., mediate Dkk activity such as its interaction with LRP5,HBM or LRP6). The polypeptides of the present invention include thoseencoded by a nucleic acid molecule with silent mutations, as well asthose nucleic acids encoding a biologically active protein withconservative amino acid substitutions, allelic variants, and othervariants of the disclosed polypeptides which maintain at least one Dkkactivity.

[0308] The amino acid compounds of the invention are polypeptides whichare partially defined in terms of amino acid residues of designatedclasses. Polypeptide homologs would include conservative amino acidsubstitutions within the amino acid classes described below. Amino acidresidues can be generally sub-classified into four major subclasses asfollows:

[0309] Acidic: The residue has a negative charge due to loss of H⁺ ionat physiological pH, and the residue is attracted by aqueous solution soas to seek the surface positions in the conformation of a peptide inwhich it is contained when the peptide is in aqueous medium, atphysiological pH.

[0310] Basic: The residue has a positive charge due to association withH⁺ ion at physiological pH, and the residue is attracted by aqueoussolution so as to seek the surface positions in the conformation of apeptide in which it is contained when the peptide is in aqueous mediumat physiological pH.

[0311] Neutral/non-polar: The residues are not charged at physiologicalpH, but the residue is repelled by aqueous solution so as to seek theinner positions in the conformation of a peptide in which it iscontained when the peptide is in aqueous medium. These residues are alsodesignated “hydrophobic.”

[0312] Neutral/polar: The residues are not charged at physiological pH,but the residue is attracted by aqueous solution so as to seek the outerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium.

[0313] It is understood, of course, that in a statistical collection ofindividual residue molecules some molecules will be charged, and somenot, and there will be an attraction for or repulsion from an aqueousmedium to a greater or lesser extent. To fit the definition of“charged”, a significant percentage (at least approximately 25%) of theindividual molecules are charged at physiological pH. The degree ofattraction or repulsion required for classification as polar or nonpolaris arbitrary and, therefore, amino acids specifically contemplated bythe invention have been classified as one or the other. Most amino acidsnot specifically named can be classified on the basis of known behavior.

[0314] Amino acid residues can be further subclassified as cyclic ornoncyclic, and aromatic or non-aromatic, self-explanatoryclassifications with respect to the side chain substituent groups of theresidues, and as small or large. The residue is considered small if itcontains a total of 4 carbon atoms or less, inclusive of the carboxylcarbon. Small residues are, of course, always nonaromatic.

[0315] The gene-encoded secondary amino acid proline, althoughtechnically within the group neutral/nonpolar/large/cyclic andnonaromatic, is a special case due to its known effects on the secondaryconformation of peptide chains, and is not, therefore, included in thisdefined group.

[0316] Other amino acid substitutions of those encoded in the gene canalso be included in peptide compounds within the scope of the inventionand can be classified within this general scheme according to theirstructure.

[0317] All of the compounds of the invention may be in the form of thepharmaceutically acceptable salts or esters. Salts may be, for example,Na⁺, K⁺, Ca⁺², Mg⁺² and the like; the esters are generally those ofalcohols of 1-6 carbons.

[0318] The present invention further provides methods for producing aprotein of the invention using nucleic acid molecules herein described.In general terms, the production of a recombinant form of a proteintypically involves the following steps.

[0319] First, a nucleic acid molecule is obtained that encodes Dkk, suchas a nucleic acid molecule encoding human Dkk or any other Dkk sequence,or that encodes a Dkk binding protein, a Dkk aptamer or a biologicallyactive fragment thereof. Particularly for Dkk binding peptides, thenucleotides encoding the peptide are incorporated into a nucleic acid inthe form of an in-frame fusion, insertion into or appended to athioredoxin coding sequence. The coding sequence (ORF) is directlysuitable for expression in any host, as it is not interrupted byintrons.

[0320] These DNAs can be transfected into host cells such as eukaryoticcells or prokaryotic cells. Eukaryotic hosts include mammalian cells andvertebrate (e.g., osteoblasts, osteosarcoma cell lines, Drosophila S2cells, hepatocytes, tumor cell lines and other bone cells of any mammal,as well as insect cells, such as Sf9 cells using recombinantbaculovirus). For example, a DNA expressing an open reading frame (ORF)under control of a type I collagen promoter, or such osteoblastpromoters as osteocalcin histone, type I collagen, TGFβ1, MSX2,cfos/cJun and Cbfal, can be used to regulate the Dkk in animal cells.Alternatively, the nucleic acid can be placed downstream from aninducible promoter, which can then be placed into vertebrate orinvertebrate cells or be used in creating a transgenic animal model.

[0321] Alternatively, proteins and polypeptides of the present inventioncan be expressed in an heterologous system. The human cell line GM637,SV40 transformed human fibroblasts, can be transfected, with a plasmidcontaining a Dkk ligand binding domain coding sequence under the controlof the chicken actin promoter (Reis et al., EMBO J. 11: 185-193 (1992)).Such transfected cells could be used as a source of Dkk binding domainin functional assays. Alternatively, polypeptides encoding only aportion of Dkk or any of the disclosed Dkk binding peptides Dkk aptamersor a polypeptide encoding a Dkk interacting protein can be expressedalone or in the form of a fusion protein. For example, Dkk derivedpeptides can be expressed in bacteria (e.g., E. coli) as GST- or His-Tagfusion proteins. These fusion proteins are then purified and can be usedto generate polyclonal antibodies or can be used to identify other Dkkligands.

[0322] The nucleic acid coding sequence is preferably placed in operablelinkage with suitable control sequences, as described above, to form anexpression unit containing the protein encoding open reading frame. Theexpression unit is used to transform a suitable host and the transformedhost is cultured under conditions that allow the production of therecombinant protein. Optionally the recombinant protein is isolated fromthe medium or from the cells; recovery and purification of the proteinmay not be necessary in some instances where some impurities may betolerated.

[0323] Each of the foregoing steps can be done in a variety of ways. Forexample, the desired coding sequences may be obtained from genomicfragments and used directly in appropriate hosts. The construction ofexpression vectors that are operable in a variety of hosts isaccomplished using appropriate replicons and control sequences, as setforth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene and were discussed in detail earlier. Suitablerestriction sites can, if not normally available, be added to the endsof the coding sequence so as to provide an excisable gene to insert intothese vectors. A skilled artisan can readily adapt any host/expressionsystem known in the art for use with the nucleic acid molecules of theinvention to produce recombinant protein.

[0324] 10. Methods to Identify Binding Partners

[0325] Another embodiment of the present invention provides methods foruse in isolating and identifying binding partners of Dkk or Dkkinteracting proteins. Dkk or a Dkk interacting protein or a polypeptidefragment thereof can be mixed with a potential binding partner or anextract or fraction of a cell under conditions that allow theassociation of potential binding partners with Dkk or with Dkkinteracting proteins. After mixing, the peptides, polypeptides, proteinsor other molecules that have become associated with Dkk or a Dkkinteracting protein are separated from the mixture. The binding partnerthat bound to the polypeptide then can be purified and further analyzed.Determination of binding partners of Dkk and Dkk interacting proteins aswell as agents which prevent the interaction of Dkk with one of itsinteracting proteins (e.g., LRP5, LRP6, HBM, or those proteins listed inFIG. 5) can be performed using a variety of different competition assaysas are known in the art. For example, the minimal sequence of Dkk, asdescribed herein, can be used to identify antibodies which compete withLRP5 (or LRP6, HBM or other ligand binding partners) for binding toDkk-1 and vice versa. The minimal Dkk sequence can be bound to thebottom of a 96-well plate (or other solid substrate), and antibodies orother potential binding agents (e.g., polypeptides, mimetics, homologs,antibody fragments and the like) can be screened in a competition assayto identify agents with binding affinities, for example, greater thanthe natural ligand binding partner of Dkk.

[0326] In the present invention, suitable cells are used for preparingassays, for the expression of a LRP and/or Dkk or proteins that interacttherewith. The cells may be made or derived from mammals, yeast, fungi,or viruses. A suitable cell for the purposes of this invention is onethat includes but is not limited to a cell that can exhibit a detectableDkk-LRP (or HBM) interaction, and preferably, the differentialinteraction between Dkk-1-LRP5 and Dkk-1-HBM. For the desired assay, thecell type may vary. In several embodiments, bone cells are preferred,for example, a human osteoblast cell (e.g. hOB-03-CE6) or osteosarcomacell (e.g. U2OS). Additional hOB cells are hOB-03-C5, hOB-02-02 and, animmortalized pre-osteocytic cell line referred to as hOB-01-C1-PS-09cells (which are deposited with American Type Culture Collection inManassas, Va. with the designation PTA-785), Examples of osteosarcomacells would include SaoS2, MG63 and HOS TE85 Immortalized refers to asubstantially continuous and permanently established cell culture withsubstantially unlimited cell division potential. That is, the cells canbe cultured substantially indefinitely, i.e., for at least about 6months under rapid conditions of growth, preferably much longer underslower growth conditions, and can be propagated rapidly and continuallyusing routine cell culture techniques. Alternatively stated, preferredcells can be cultured for at least about 100, 150 or 200 populationdoublings. These cells produce a complement of proteins characteristicof normal human osteoblastic cells and are capable of osteoblasticdifferentiation. They can be used in cell culture studies ofosteoblastic cell sensitivity to various agents, such as hormones,cytokines, and growth factors, or in tissue therapy. Certain non bonecells such as HEK 293 cells that exhibit detectable Dkk-LRP (or HBM)interaction are also be useful for the assays of this invention.

[0327] To identify and isolate a binding partner, the entire Dkk protein(e.g., human Dkk-1, GenBank Accession No. BM34651) or a Dkk interactingprotein (Genbank Accession Nos. for some Dkk-1 interacting proteins aregiven in FIG. 5) can be used. Alternatively, a polypeptide fragment ofthe protein can be used. Suitable fragments of the protein include atleast about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150 or morecontiguous amino acid residues of any Dkk or Dkk interactor sequence.Preferable sequences of Dkk include portions or all of one or both ofthe cysteine rich domains (e.g., Cys-1 and Cys-2 of Dkk-1) or theconserved sequences at the amino terminus of Dkk-1 (See Krupnik et al.,Gene 238: 301-313 (1999)). Alternatively, portions of LRP5, LRP6, HBMand other Dkk interacting proteins such as those in FIG. 5 that interactwith Dkk-1 can be used to identify and isolate agents which modulate Dkkactivity. Alternatively, peptide aptamers of LRP5, LRP6, HBM, Dkk andother Dkk interacting proteins such as those in FIG. 5 that interactwith Dkk-1 can be used to identify and isolate agents which modulate Dkkactivity.

[0328] As used herein, a cellular extract refers to a preparation orfraction which is made from a lysed or disrupted cell. A variety ofmethods can be used to obtain cell extracts. Cells can be disruptedusing either physical or chemical disruption methods. Examples ofphysical disruption methods include, but are not limited to, sonicationand mechanical shearing. Examples of chemical lysis methods include, butare not limited to, detergent lysis and enzyme lysis. A skilled artisancan readily adapt methods for preparing cellular extracts in order toobtain extracts for use in the present methods.

[0329] Once an extract of a cell is prepared, the extract is mixed withthe protein of the invention under conditions in which association ofthe protein with the binding partner can occur. A variety of conditionscan be used, the most preferred being conditions that closely resembleconditions found in the cytoplasm of a human cell. Features such asosmolarity, pH, temperature, and the concentration of cellular extractused, can be varied to optimize the association of the protein with thebinding partner.

[0330] After mixing under appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be utilized toseparate the mixture. For example, antibodies specific to a protein ofthe invention can be used to immunoprecipitate the binding partnercomplex. Alternatively, standard chemical separation techniques such aschromatography and density/sediment centrifugation can be used. Forexample, a protein of the invention is expressed with an affinity tagsuch as a His tag. The His labeled protein and any bound molecule may beretained and selectively eluted from a Ni-NTA column.

[0331] After removal of non-associated cellular constituents found inthe extract, the binding partner can be dissociated from the complexusing conventional methods. For example, dissociation can beaccomplished by altering the salt concentration or pH of the mixture.

[0332] To aid in separating associated binding partner pairs from themixed extract, the protein of the invention can be immobilized on asolid support. For example, the protein can be attached to anitrocellulose matrix or acrylic beads. Attachment of the protein to asolid support aids in separating peptide/binding partner pairs fromother constituents found in the extract. The identified binding partnerscan be either a single protein or a complex made up of two or moreproteins.

[0333] Alternatively, the nucleic acid molecules of the invention can beused in a Y2H system. The Y2H system has been used to identify otherprotein partner pairs and can readily be adapted to employ the nucleicacid molecules herein described. Methods of performing and using Y2Hsystems are known. See, e.g., Finley et al., “Two-Hybrid Analysis ofGenetic Regulatory Networks,” in The Yeast Two-Hybrid System (Paul L.Bartel et al., eds., Oxford, 1997); Meijia Yang, “Use of a CombinatorialPeptide Library in the Two-Hybrid Assay,” in The Yeast Two-Hybrid System(Paul L. Bartel et al., eds., Oxford, 1997); Gietz et al.,“Identification of proteins that interact with a protein of interest:Applications of the yeast two-hybrid system,” Mol. & Cell. Biochem. 172:67-9 (1997); K. H. Young, “Yeast Two-Hybrid: So Many Interactions,(in)so Little Time,” Biol. Reprod. 58: 302-311 (1998); R. Brent et al.,“Understanding Gene and Allele Function with Two-Hybrid Methods,” Annu.Rev. Genet 31:663-704 (1997) and U.S. Pat. No. 5,989,808. The Dkk-1interacting proteins identified in FIG. 5 were identified using the Y2Hinteracting system using Dkk-1 as bait.

[0334] One preferred in vitro binding assay for Dkk modulators wouldcomprise a mixture of a LRP binding domain of Dkk and one or morecandidate binding targets or substrates. After incubating the mixtureunder appropriate conditions, one would determine whether Dkk or afragment thereof bound with the candidate modulator present. Forcell-free binding assays, one or more of the components usuallycomprises or is coupled to a label. The label may provide for directdetection, such as radioactivity, luminescence, optical or electrondensity, etc., or indirect detection such as an epitope tag, an enzyme,etc. A variety of methods may be employed to detect the label dependingon the nature of the label and other assay components. For example, thelabel may be detected bound to the solid substrate or a portion of thebound complex containing the label may be separated from the solidsubstrate, and the label thereafter detected. Fluorescence resonanceenergy transfer may be utilized to monitor the interaction of twolabeled molecules. For example, a fluorescence label on Dkk and anotherlabel on LRP5 or a soluble fragment thereof such as the extracellulardomain will exchange fluorescence resonance energy when in closeproximity indicating that the two molecules are bound. A preferredbinding partner for Dkk will increase or decrease the affinity betweenDkk and LRP5 which will be readily observable in a fluorescencespectrometer. Alternatively, an instrument, such as a surface plasmonresonance detector manufactured by BlAcore (Uppsala, Sweden), may beused to observe interactions with a fixed target. One skilled in the artknows of many other methods which may be employed for this purpose.

[0335] Thereby, the present invention provides methods for screeningcandidates including polypeptides of the present invention for activitywhich identifies these candidates as valuable drug leads. Other suitablemethods are also known in the art and are suitable for use herein,including Xenopus oocyte injection studies and TCF luciferase assays.

[0336] Additional assays can be used to identify the activity of Dkk andDkk interacting proteins in the Wnt pathway, as well as the impact ofmodulators of Dkk and Dkk interacting proteins on the Wnt pathway. Theseinclude, for example, a Xenopus embryo assay and a TCF-luciferasereporter gene assay to monitor Wnt signaling modulation.

[0337] Xenopus embryos are an informative in vivo assay system toevaluate the modulation of Wnt signaling. Ectopic expression of certainWnts or other activators of the Wnt signaling pathway results in abifurcation of the anterior neural plate. This bifurcation results in aduplicated body axis, which suggests a role for Wnt signaling duringembryonic development (McMahon et al., Cell 58: 1075-84 (1989); Sokol etal., Cell 67: 741-52 (1991)). Since these original observations, theXenopus embryo assay has been extensively used as an assay system forevaluating modulation of the Wnt signaling pathway. One preferredembodiment of the present invention is demonstrated in Example 6.

[0338] Constructs for Xenopus expression can be prepared as would beknown in the art. For example, a variety of cDNAs have been engineeredinto the vector pCS2+ (Turner et al., Genes Devel. 8: 1434-1447 (1994))to facilitate the in vitro generation of mRNA for use in Xenopus embryoinjection experiments. DNA inserts are subcloned in the senseorientation with respect to the vector SP6 promoter. Downstream of theinsert, the vector provides an SV40 virus polyadenlylation signal and aT3 promoter sequence (i.e., for the generation of antisense mRNA).Constructs can be generated for various Dkk family members, LRP5, LRP6,HBM, Dkk-1 interactors, etc. Constructs could be generated in pCS2⁺ thatcontain the nucleic acid sequence encoding for the peptide aptamers thatwere identified in yeast screens. These sequences would be fused to a 5′synthetic translation initiation sequence followed by a canonical signalsequence to ensure that the peptide aptamer would be translated andsecreted from the cell.

[0339] Once these constructs are made then mRNA can be synthesized andinjected into Xenopus oocytes. mRNA for microinjection into Xenopusembryos is generated by in vitro transcription using the cDNA constructsin the pCS2⁺ vector described above as template. Various amounts of RNAcan be injected into the ventral blastomere of the 4- or 8-cell Xenopusembryo substantially as described in Moon et al., Technique-J. ofMethods in Cell and Mol. Biol. 1: 76-89 (1989), and Peng, Meth. Cell.Biol. 36: 657-62 (1991).

[0340] Previous data has shown that expression of LRP5, in the presenceof Wnt5a, results in a Wnt-induced duplicated axis formation in Xenopusembryos (Tamai et a., Nature 407: 530-535 (2000)). The roles of Dkk-1and Dkk-2, and Dkk-1 interacting proteins, in modulating theLRP5-mediated Wnt response in vivo can be analyzed using, for example,the Xenopus embryo. In addition, the peptide aptamers, Dkk interactingproteins, or combinations of the above can be evaluated in a similarmanner.

[0341] Experiments can also be conducted wherein RNA is injected intothe dorsal blastomere to ensure the specificity of the observedphenotypes. Lineage tracing experiments can be performed where a markergene such as green fluorescent protein (GFP) or LacZ is co-injected withthe experimental RNAs. Detecting marker gene expression would identifythe targeted cells of the microinjection and aid in elucidating themechanism of action. In addition to the Wnt signaling components listedabove, the point at which HBM acts upon the Wnt pathway can also beanalyzed. This can be done by co-injections of various dominant-negativeconstructs. For example, a dominant negative TCF-3 construct would beuseful to demonstrate that the observed axis duplication (and Wntactivation) is mediated via the β-catenin-TCF response. If so, such aconstruct would be expected to abolish the observed duplicated axisphenotype. Another example would include a dominant negative Dshconstruct. Since Dsh is far upstream in the Wnt signaling pathway, adominant negative construct should abolish the activation of the Wntresponse and the observed axis duplication. If it does not, this wouldsuggest that axis duplication is being induced via a different signalingpathway.

[0342] The marker genes of the injected Xenopus embryos can be analyzedas follows. Representative embryos are collected at stage 10.5 (11 hourspost fertilization) for marker gene analysis. RNA is extracted andpurified from the embryos following standard protocols (Sambrook et al.,1989 at 7.16). Marker genes could include the following: Siamois (i.e.,Wnt responsive gene), Xnr3 (i.e., Wnt responsive gene), slug (i.e.,neural crest marker), Xbra (i.e., early mesoderm marker), HNK-1 (i.e.,ectodermal/neural marker), endodermin (Le., endoderm), Xlhbox8 (i.e.,pancreatic), BMP2 and BMP4 (i.e., early mesoderm), XLRP6 (i.e., maternaland zygotic expression, it is also the LRP6 homolog in the frog), EF-1(i.e., control) and ODC (i.e., control). Induction of marker genes isanalyzed and quantitated by RT-PCR/TaqMan®.

[0343] This type of marker analysis is excellent to monitor changes ingene expression that result very early in the embryo as a direct resultof signaling perturbation. Other experiments could be designed thatwould monitor changes in gene expression in a more tissue orspatially-restricted fashion. Examples would include the generation of atransgenic Xenopus model. For example, Zmax/LRP5 and HBM expressioncould be under the control of the brachyury or cardiac-actin promotersdirecting gene expression transiently in the mesoderm or in the somites,respectively. Phenotype analyses of these transgenic Xenopus animalswould include marker gene analysis/transcriptional profiling (from arestricted tissue source) and histologic examination of the tissue.

[0344] A TCF-luciferase assay system such as that described in Example 7can also be used to monitor Wnt signaling activity, Dkk activity and Dkkinteracting protein activity. Constructs for the TCF-luciferase assayscan be prepared as would be known in the art. For example, Dkk and Dkkinteracting protein peptides, LRP5/LRP6, among others, can be expressedin pcDNA3.1, using Kozak and signal sequences to target peptides forsecretion.

[0345] Once constructs have been prepared, cells such as osteoblasts andHEK293 cells are seeded in well plates and transfected with constructDNA, CMV beta-galactosidase plasmid DNA, and TCF-luciferase reporterDNA. The cells are then lysed and assayed for beta-galactosidase andluciferase activity to determine whether Dkk, Dkk interacting proteins,or other molecules such as antibodies affect Wnt signaling.

[0346] Additional assays for monitoring Wnt signaling activity, Dkkactivity, and Dkk interacting protein activity include:

[0347] Modulation of another Wnt-responsive transcription factor, LEF,as visualized by a reporter gene activity. One example includes theactivation of the LEF1 promoter region fused to the luciferase reportergene (Hsu et al., Mol. Cell. Biol. 18: 4807-18 (1999)).

[0348] Alterations in cell proliferation, cell cycle or apoptosis. Thereare numerous examples describing Wnt-mediated cellular transformationsincluding Shimizu et al., Cell. Growth Differ. 8: 1349-58 (1997).

[0349] Stabilization and cellular localization of de-phosphorylatedβ-catenin as an indicator of Wnt activation (Shimizu et al., 1997).

[0350] Additional methods of assaying Wnt signaling, through either thecanonical or non-canonical pathways, would be apparent to the artisan ofordinary skill.

[0351] 11. Methods to Identify Agents that Modulate the Expression of aNucleic Acid Encoding the Dkk and/or LRP5 Proteins and/or DkkInteracting Proteins

[0352] Another embodiment of the present invention provides methods foridentifying agents that modulate the expression of a nucleic acidencoding Dkk. Such assays may utilize any available means of monitoringfor changes in the expression level of the nucleic acids of theinvention. As used herein, an agent is said to modulate the expressionof Dkk, if it is capable of up- or down-regulating expression of thenucleic acid in a cell (e.g., mRNA).

[0353] In one assay format, cell lines that contain reporter genefusions between the nucleic acid encoding Dkk (or proteins whichmodulate the activity of Dkk) and any assayable fusion partner may beprepared. Numerous assayable fusion partners are known and readilyavailable, including but not limited to the firefly luciferase gene andthe gene encoding chloramphenicol acetyltransferase (Alam et al., Anal.Biochem. 188: 245-254 (1990)). Cell lines containing the reporter genefusions are then exposed to the agent to be tested under appropriateconditions and time. Differential expression of the reporter genebetween samples exposed to the agent and control samples identifiesagents which modulate the expression of a nucleic acid encoding Dkk orother protein which modulates Dkk activity. Such assays can similarly beused to determine whether LRP5 and even LRP6 activity is modulated byregulating Dkk activity.

[0354] Additional assay formats may be used to monitor the ability ofthe agent(s) to modulate the expression of a nucleic acid encoding Dkk,alone or Dkk and LRP5, and/or Dkk interacting proteins such as thoseidentified in FIG. 5. For instance, mRNA expression may be monitoreddirectly by hybridization to the nucleic acids of the invention. Celllines are exposed to the agent to be tested under appropriate conditionsand time and total RNA or mRNA is isolated by standard procedures suchthose disclosed in Sambrook et al. (1989); Ausubel et al., CurrentProtocols in Molecular Biology (Greene Publishing Co., NY, 1995);Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1982); and Short Protocolsin Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology (Frederick M. Ausubel et al., April 1999).

[0355] Probes to detect differences in RNA expression levels betweencells exposed to the agent and control cells may be prepared from thenucleic acids of the invention. It is preferable, but not necessary, todesign probes which hybridize only with target nucleic acids underconditions of high stringency. Only highly complementary nucleic acidhybrids form under conditions of high stringency. Accordingly, thestringency of the assay conditions determines the amount ofcomplementarity which should exist between two nucleic acid strands inorder to form a hybrid. Stringency should be chosen to maximize thedifference in stability between the probe:target hybrid and potentialprobe:non-target hybrids.

[0356] Probes may be designed from the nucleic acids of the inventionthrough methods known in the art. For instance, the G+C content of theprobe and the probe length can affect probe binding to its targetsequence. Methods to optimize probe specificity are commonly available.See for example, Sambrook et al. (1989) or Ausubel et al. (CurrentProtocols in Molecular Biology, Greene Publishing Co., NY, 1995).

[0357] Hybridization conditions are modified using known methods, suchas those described by Sambrook et al. (1989) and Ausubel et al. (1995),as suitable for each probe. Hybridization of total cellular RNA or RNAenriched for polyA RNA can be accomplished in any available format. Forinstance, total cellular RNA or RNA enriched for polyA RNA can beaffixed to a solid support and the solid support exposed to at least oneprobe comprising at least one, or part of one of the nucleic acidsequences of the invention under conditions in which the probe willspecifically hybridize. Alternatively, nucleic acid fragments comprisingat least one, or part of one of the sequences of the invention can beaffixed to a solid support, such as a porous glass wafer. The glass orsilica wafer can then be exposed to total cellular RNA or polyA RNA froma sample under conditions in which the affixed sequences willspecifically hybridize. Such glass wafers and hybridization methods arewidely available, for example, those disclosed by Beattie (WO 95/11755).By examining for the ability of a given probe to specifically hybridizeto an RNA sample from an untreated cell population and from a cellpopulation exposed to the agent, agents which up- or down-regulate theexpression of a nucleic acid encoding Dkk, a Dkk interacting protein,and/or LRP5 can be identified.

[0358] Microarray technology and transcriptional profiling are examplesof methods which can be used to analyze the impact of putative Dkk orDkk interacting protein modulating compounds. For transcriptionalprofiling, mRNA from cells exposed in vivo to a potential Dkk modulatingagent, such as the Dkk interacting proteins identified in the presentinvention (e.g., those identified in FIG. 5), agents which modulate Dkkinteracting proteins, and mRNA from the same type of cells that were notexposed to the agent could be reverse transcribed and hybridized to achip containing DNA from numerous genes, to thereby compare theexpression of genes in cells treated and not treated with the agent. If,for example a putative Dkk modulating agent down-regulates theexpression of Dkk in the cells, then use of the agent may be undesirablein certain patient populations. For additional methods oftranscriptional profiling and the use of microarrays, refer to, forexample, U.S. Pat. No. 6,124,120 issued to Lizardi (2000).

[0359] Additional methods for screening the impact of Dkk and Dkkinteracting protein modulating compounds or the impact of Dkk or Dkkinteracting proteins on modulation of LRP5, LRP6, HBM or the Wnt pathwayinclude the use of TaqMan PCR, conventional reverse transcriptase PCR(RT-PCR), changes in downstream surrogate markers (i.e., Wnt responsivegenes), and anti-Dkk Western blots for protein detection. Other methodswould be readily apparent to the artisan of ordinary skill.

[0360] 12. Methods to Identify Agents that Modulate at Least OneActivity of Dkk, a Dkk Interacting Protein, or LRP5/LRP6/HBM

[0361] Another embodiment of the present invention provides methods foridentifying agents that modulate at least one activity of Dkk, Dkkinteracting proteins, and/or LRP5/LRP6/HBM proteins or preferably whichspecifically modulate an activity of a Dkk/Dkk interacting proteincomplex or an LRP5(or LRP6/HBM)/Dkk complex, or a biologically activefragment of Dkk (e.g., comprising the domain which binds LRP5/LRP6/HBM)or a Dkk interacting protein complex. Such methods or assays may utilizeany means of monitoring or detecting the desired activity as would beknown in the art (See, e.g., Wu et al., Curr. Biol. 10:1611-4 (2000);Fedi et al., J. Biol. Chem. 274:19465-72 (1991); Grotewold et al., Mech.Dev. 89:151-3 (1999); Shibata et al., Mech. Dev. 96:243-6 (2000); Wanget al., Oncogene 19:1843-8 (2000); and Glinka et al., Nature 391:357-62(1998)). Potential agents which modulate Dkk include, for example, p53,the tumor suppressor protein, which can induce Dkk-1. Damage to DNA hasalso been observed to up-regulate Dkk-1 expression via a stabilizationand activation of p53 (Wang et al., Oncogene 19:1843-48 (2000)); and,Shou et al., Oncogene 21:878-89 (2002)). Additionally, Fedi et al.(1999) purportedly showed that Dkk-1 can block the Wnt2-inducedoncogenic transformation of NIH-3T3 cells. Furthermore, it has beensuggested that Dkk expression can be modulated by BMP signaling in thedeveloping skeleton (Mukhopadhyay et al., Dev. Cell. 1:423-34 (2001);and Grotewold et al., EMBO J. 21:966-75 (2002)). Grotewold et al.additionally describe altered Dkk expression levels in response tostress signals including UV irradiation and other genotoxic stimuli.They propose that Dkk expression is proapoptotic. In animals expressingHBM constructs conferring high bone mass, a reduced osteoblast apoptosiseffect was observed. Thus, HBM and HBM-like variants may control/alterDkk's role in programmed cell death. Other agents which potentiallymodulate Dkk activity include the Dkk interacting proteins identified inFIG. 5.

[0362] In one embodiment, the relative amounts of Dkk or a Dkkinteracting protein of a cell population that has been exposed to theagent to be tested is compared to an unexposed control cell population.Antibodies can be used to monitor the differential expression of theprotein in the different cell populations. Cell lines or populations areexposed to the agent to be tested under appropriate conditions and time.Cellular lysates may be prepared from the exposed cell line orpopulation and a control, unexposed cell line or population. Thecellular lysates are then analyzed with the probe, as would be known inthe art. See, e.g., Ed Harlow and David Lane, Antibodies: A LaboratoryManual (Cold Spring Harbor, N.Y., 1988) and Ed Harlow and David Lane,Using Antibodies: A Laboratory Manual (Cold Spring Harbor, N.Y. 1998).

[0363] For example, N- and C- terminal fragments of Dkk can be expressedin bacteria and used to search for proteins which bind to thesefragments. Fusion proteins, such as His-tag or GST fusion to the N- orC-terminal regions of Dkk (or to biologically active domains of Dkk-1)or a whole Dkk protein can be prepared. These fusion proteins can becoupled to, for example, Talon or Glutathione-Sepharose beads and thenprobed with cell lysates to identify molecules which bind to Dkk. Priorto lysis, the cells may be treated with purified Wnt proteins, RNA, ordrugs which may modulate Wnt signaling or proteins that interact withdownstream elements of the Wnt pathway. Lysate proteins binding to thefusion proteins can be resolved by SDS-PAGE, isolated and identified by,for example protein sequencing or mass spectroscopy, as is known in theart. See, e.g., Protein Purification Applications: A Practical Approach(Simon Roe, ed., 2^(nd) ed. Oxford Univ. Press, 2001) and “Guide toProtein Purification” in Meth. Enzymology vol. 182 (Academic Press,1997).

[0364] The activity of Dkk, a Dkk interacting protein, or a complex ofDkk with LRP5/LRP6/HBM may be affected by compounds which modulate theinteraction between Dkk and a Dkk interacting protein (such as thoseshown in FIG. 5) and/or Dkk and LRP5/LRP6/HBM. The present inventionprovides methods and research tools for the discovery andcharacterization of these compounds. The interaction between Dkk and aDkk interacting protein and/or Dkk and LRP5/6/HBM may be monitored invivo and in vitro. Compounds which modulate the stability of aDkk-LRP5/LRP6/HBM complex are potential therapeutic compounds. Examplein vitro methods include: Binding LRP5/6/HBM, Dkk, or a Dkk interactingprotein to a sensor chip designed for an instrument such are made byBiacore (Uppsala, Sweden) for the performance of an plasmon resonancespectroscopy observation. In this method, the chip with one of Dkk, aDkk interacting protein, or LRP5/6 is first exposed to the other underconditions which permit them to form the complex. A test compound isthen introduced and the output signal of the instrument provides anindication of any effect exerted by the test compound. By this method,compounds may be rapidly screened. Another, in vitro, method isexemplified by the SAR-by-NMR methods (Shuker et al., Science.274:1531-4 (1996)). Briefly, a Dkk-1 binding domain and/or LRP 5 or 6LBD are expressed and purified as ¹⁵N labeled protein by expression inlabeled media. The labeled protein(s) are allowed to form the complex insolution in an NMR sample tube. The heteronuclear correlation spectrumin the presence and absence of a test compound provides data at thelevel of individual residues with regard to interactions with the testcompound and changes at the protein-protein interface of the complex.One of skill in the art knows of many other protocols, e.g. affinitycapillary electrophoresis (Okun et al. J Biol Chem 276:1057-62 (2001);Vergun and Chu, Methods, 19:270-7 (1999)), fluorescence spectroscopy,electron paramagnetic resonance, etc. which can monitor the modulationof a complex and/or measure binding affinities for complex formation.

[0365] In vitro protocols for monitoring the modulation of aDkk/LRP5/LRP6/HBM complex include the yeast two hybrid protocol. Theyeast two hybrid method may be used to monitor the modulation of acomplex in vivo by monitoring the expression of genes activated by theformation of a complex of fusion proteins of Dkk and LRP ligand bindingdomains. Nucleic acids according to the invention which encode theinteracting Dkk and LRP LBD domains are incorporated into bait and preyplasmids. The Y2H protocol is performed in the presence of one or moretest compounds. The modulation of the complex is observed by a change inexpression of the complex activated gene. It will be appreciated by oneskilled in the art that test compounds can be added to the assaydirectly or, in the case of proteins, can be coexpressed in the yeastwith the bait and prey compounds. Similarly, fusion proteins of Dkk andDkk interacting proteins can also be used in a Y2H screen to identifyother proteins which modulate the Dkk/Dkk interacting protein complex.

[0366] Assay protocols such as these may be used in methods to screenfor compounds, drugs, treatments which modulate the Dkk/Dkk interactingprotein and/or Dkk/LRP5/6 complex, whether such modulation occurs bycompetitive binding, or by altering the structure of either LRP 5/6 orDkk at the binding site, or by stabilizing or destablizing theprotein-protein interface. It may be anticipated that peptide aptamersmay competitively bind, although induction of an altered binding sitestructure by steric effects is also possible.

[0367] 12.1 Antibodies and Antibody Fragments

[0368] Polyclonal and monoclonal antibodies and fragments of theseantibodies which bind to Dkk or LRP5/LRP6/HBM can be prepared as wouldbe known in the art. For example, suitable host animals can be immunizedusing appropriate immunization protocols and the peptides, polypeptidesor proteins of the invention. Peptides for use in immunization aretypically about 8-40 residues long. If necessary or desired, thepolypeptide immunogens can be conjugated to suitable carriers. Methodsfor preparing immunogenic conjugates with carriers such as bovine serumalbumin (BSA), keyhole limpet hemocyanin (KLH), or other carrierproteins are well known in the art (See, Harlow et al., 1988). In somecircumstances, direct conjugation using, for example, carbodiimidereagents, may be effective; in other instances linking reagents such asthose supplied by Pierce Chemical Co., Rockford, Ill., may be desirableto provide accessibility to the polypeptide or hapten. The haptenpeptides can be extended at either the amino or carboxy terminus with acysteine residue or interspersed with cysteine residues, for example, tofacilitate linking to a carrier. Administration of the immunogens isconducted generally by injection over a suitable time period and withuse of suitable adjuvants, as is generally understood in the art. Duringthe immunization schedule, titers of antibodies are taken to determineadequacy of antibody formation.

[0369] Anti-peptide antibodies can be generated using syntheticpeptides, for example, the peptides derived from the sequence of anyDkk, including Dkk-1, or LRP5/LRP6/HBM. Synthetic peptides can be assmall as 2-3 amino acids in length, but are preferably at least 3, 5,10, or 15 or more amino acid residues long. Such peptides can bedetermined using programs such as DNAStar. The peptides are coupled toKLH using standard methods and can be immunized into animals such asrabbits. Polyclonal anti-Dkk or anti-LRP5/LRP6/HBM peptide antibodiescan then be purified, for example using Actigel beads containing thecovalently bound peptide.

[0370] While the polyclonal antisera produced in this way may besatisfactory for some applications, for pharmaceutical compositions, useof monoclonal preparations is preferred. Immortalized cell lines whichsecrete the desired monoclonal antibodies may be prepared using thestandard method of Kohler and Milstein or modifications which effectimmortalization of lymphocytes or spleen cells, as is generally known(See, e.g., Harlow et al., 1988 and 1998). The immortalized cell linessecreting the desired antibodies can be screened by immunoassay in whichthe antigen is the peptide hapten, polypeptide or protein. When theappropriate immortalized cell culture secreting the desired antibody isidentified, the cells can be cultured either in vitro or by productionin ascites fluid.

[0371] The desired monoclonal antibodies are then recovered from theculture supernatant or from the ascites supernatant. Fragments of themonoclonal antibodies which contain the immunologically significantportion can be used as agonists or antagonists of Dkk activity. Use ofimmunologically reactive fragments, such as the Fab, scFV, Fab′, ofF(ab′)₂ fragments are often preferable, especially in a therapeuticcontext, as these fragments are generally less immunogenic than thewhole immunoglobulin.

[0372] The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of Dkk or LRP5/LRP6/HBM can also be produced in thecontext of chimeras with multiple species origin. Antibody reagents socreated are contemplated for use diagnostically or as stimulants orinhibitors of Dkk activity.

[0373] In one embodiment, antibodies against Dkk, bind Dkk with highaffinity, i.e., ranging from 10⁻⁵ to 10⁻⁹ M. Preferably, the anti-Dkkantibody will comprise a chimeric, primate, Primatized®, human orhumanized antibody. Also, the invention embraces the use of antibodyfragments, e.g., Fab's, Fv's, Fab's, F(ab)₂, and aggregates thereof.

[0374] Another embodiment contemplates chimeric antibodies whichrecognize Dkk or LRP5/LRP6/HBM. A chimeric antibody is intended to referto an antibody with non-human variable regions and human constantregions, most typically rodent variable regions and human constantregions.

[0375] A “primatized® antibody” refers to an antibody with primatevariable regions, e.g., CDR's, and human constant regions. Preferably,such primate variable regions are derived from an Old World monkey.

[0376] A “humanized antibody” refers to an antibody with substantiallyhuman framework and constant regions, and non-humancomplementarity-determining regions (CDRs). “Substantially” refers tothe fact that humanized antibodies typically retain at least severaldonor framework residues (i.e., of non-human parent antibody from whichCDRs are derived).

[0377] Methods for producing chimeric, primate, primatized®, humanizedand human antibodies are well known in the art. See, e.g., U.S. Pat. No.5,530,101, issued to Queen et al.; U.S. Pat. No. 5,225,539, issued toWinter et al.; U.S. Pat. Nos. 4,816,397 and 4,816,567, issued to Boss etal. and Cabilly et al. respectively, all of which are incorporated byreference in their entirety.

[0378] The selection of human constant regions may be significant to thetherapeutic efficacy of the subject anti-Dkk or LRP5/LRP6/HBM antibody.In a preferred embodiment, the subject anti-Dkk or LRP5/LRP6/HBMantibody will comprise human, gamma 1, or gamma 3 constant regions and,more preferably, human gamma 1 constant regions.

[0379] Methods for making human antibodies are also known and include,by way of example, production in SCID mice, and in vitro immunization.

[0380] The subject anti-Dkk or LRP5/LRP6/HBM antibodies can beadministered by various routes of administration, typically parenteral.This is intended to include intravenous, intramuscular, subcutaneous,rectal, vaginal, and administration with intravenous infusion beingpreferred.

[0381] The anti-Dkk or LRP5/LRP6/HBM antibody will be formulated fortherapeutic usage by standard methods, e.g., by addition ofpharmaceutically acceptable buffers, e.g., sterile saline, sterilebuffered water, propylene glycol, and combinations thereof.

[0382] Effective dosages will depend on the specific antibody, conditionof the patient, age, weight, or any other treatments, among otherfactors. Typically effective dosages will range from about 0.001 toabout 30 mg/kg body weight, more preferably from about 0.01 to 25 mg/kgbody weight, and most preferably from about 0.1 to about 20 mg/kg bodyweight.

[0383] Such administration may be effected by various protocols, e.g.,weekly, bi-weekly, or monthly, depending on the dosage administered andpatient response. Also, it may be desirable to combine suchadministration with other treatments.

[0384] Antibodies to Dkk-1 interacting proteins, such as thoseidentified in FIG. 5, are also contemplated according to the presentinvention, and can be used similarly to the Dkk-1 antibodies mentionedin the above methodology.

[0385] The antibodies of the present invention can be utilized inexperimental screening, as diagnostic reagents, and in therapeuticcompositions.

[0386] 12.2 Chemical Libraries

[0387] Agents that are assayed by these methods can be randomly selectedor rationally selected or designed. As used herein, an agent is said tobe randomly selected when the agent is chosen randomly withoutconsidering the specific sequences involved in the association of Dkk-1alone, Dkk-1 interacting proteins alone, or with their associatedsubstrates, binding partners, etc. An example of randomly selectedagents is the use of a chemical library or a peptide combinatoriallibrary, or a growth broth of an organism.

[0388] The agents of the present invention can be, as examples,peptides, small molecules, vitamin derivatives, as well ascarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents of the presentinvention.

[0389] 12.3 Peptide Synthesis

[0390] The peptide agents of the invention can be prepared usingstandard solid phase (or solution phase) peptide synthesis methods, asis known in the art. In addition, the DNA encoding these peptides may besynthesized using commercially available oligonucleotide synthesisinstrumentation and produced recombinantly using standard recombinantproduction systems. The production of polypeptides using solid phasepeptide synthesis is necessitated if non-nucleic acid-encoded aminoacids are to be included.

[0391] 13. Uses for Agents that Modulate at Least One Activity of Dkk. aDkk Interacting Protein, a Dkk/Dkk Interacting Protein Complex, or aDkk/LRP5 or Dkk/LRP6 Complex

[0392] The proteins and nucleic acids of the invention, such as theproteins or polypeptides containing an amino acid sequence of LRP5, Dkk,and Dkk interacting proteins are involved in bone mass modulation andlipid modulation of other Wnt pathway mediated activity. Agents thatmodulate (i.e., up and down-regulate) the expression of Dkk or Dkkinteracting proteins, or agents, such as agonists and antagonistsrespectively, of at least one activity of Dkk or a Dkk interactingprotein may be used to modulate biological and pathologic processesassociated with the function and activity of Dkk or a Dkk interactingprotein.

[0393] As used herein, a subject can be preferably any mammal, so longas the mammal is in need of modulation of a pathological or biologicalprocess modulated by a protein of the invention. The term “mammal” meansan individual belonging to the class Mammalia. The invention isparticularly useful in the treatment of human subjects.

[0394] As used herein, a biological or pathological process modulated byDkk or a Dkk interacting protein may include binding of Dkk to a Dkkinteracting protein, Dkk to LRP5 or LRP6 or release therefrom,inhibiting or activating Dkk or a Dkk interacting protein mRNA synthesisor inhibiting Dkk or Dkk interacting protein modulated inhibition ofLRP5 or LRP6 mediated Wnt signaling. Further bone-related markers may beobserved such as alkaline phosphatase activity, osteocalcin production,or mineralization.

[0395] Pathological processes refer to a category of biologicalprocesses which produce a deleterious effect. For example, expression orup-regulation of expression of LRP5 or LRP6 and/or Dkk and/or a Dkkinteracting protein may be associated with certain diseases orpathological conditions. As used herein, an agent is said to modulate apathological process when the agent statistically significantly (p<0.05)alters the process from its base level in the subject. For example, theagent may reduce the degree or severity of the process mediated by thatprotein in the subject to which the agent was administered. Forinstance, a disease or pathological condition may be prevented, ordisease progression modulated by the administration of agents whichreduce or modulate in some way the expression or at least one activityof a protein of the invention.

[0396] As LRP5/6 and Dkk are involved both directly and indirectly inbone mass modulation, one embodiment of this invention is to use Dkk orDkk interacting protein expression as a method of diagnosing a bonecondition or disease. Certain markers are associated with specific Wntsignaling conditions (e.g., TCF/LEF activation). Diagnostic tests forbone conditions may include the steps of testing a sample or an extractthereof for the presence of Dkk or Dkk interacting protein nucleic acids(i.e., DNA or RNA), oligomers or fragments thereof or protein productsof TCF/LEF regulated expression. For example, standard in situhybridization or other imaging techniques can be utilized to observeproducts of Wnt signaling.

[0397] This invention also relates to methods of modulating bonedevelopment or bone loss conditions. Inhibition of bone loss may beachieved by inhibiting or modulating changes in the LRP5/6 mediated Wntsignaling pathway. For example, absence of LRP5 activity may beassociated with low bone mass. Increased activity LRP5 may be associatedwith high bone mass. Therefore, modulation of LRP5 activity will in turnmodulate bone development. Modulation of the Dkk1 LRP5/6 or Dkk/Dkkinteracting protein complex via agonists and antagonists is oneembodiment of a method to regulate bone development. Such modulation ofbone development can result from inhibition of the activity of, forexample, a Dkk/LRP(5/6) protein complex, a Dkk/Dkk interacting proteincomplex, upregulated transcription of the LRP5 gene or inhibitedtranslation of Dkk or Dkk interacting protein mRNA.

[0398] The agents of the present invention can be provided alone, or incombination with other agents that modulate a particular pathologicalprocess. As used herein, two agents are said to be administered incombination when the two agents are administered simultaneously or areadministered independently in a fashion such that the agents will act atthe same time.

[0399] The agents of the present invention can be administered viaparenteral, subcutaneous (sc), intravenous (iv), intramuscular (im),intraperitoneal (ip), transdermal or buccal routes. Alternatively, orconcurrently, administration may be by the oral route. The dosageadministered will be dependent upon the age, health, and weight of therecipient, kind of concurrent treatment, if any, frequency of treatment,and the nature of the effect desired.

[0400] The present invention further provides compositions containingone or more agents which modulate expression or at least one activity ofa protein of the invention. While individual needs vary, determinationof optimal ranges of effective amounts of each component is within theskill of the art. Typical dosages of the active agent which mediate Dkkor Dkk interacting protein activity comprise from about 0.0001 to about50 mg/kg body weight. The preferred dosages comprise from about 0.001 toabout 50 mg/kg body weight. The most preferred dosages comprise fromabout 0.1 to about 1 mg/kg body weight. In an average human of 70 kg,the range would be from about 7 pg to about 3.5 g, with a preferredrange of about 0.5 mg to about 5 mg.

[0401] In addition to the pharmacologically active agent, thecompositions of the present invention may contain suitablepharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically for delivery to the siteof action. Suitable formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form, forexample, water-soluble salts. In addition, suspensions of the activecompounds as appropriate oily injection suspensions may be administered.Suitable lipophilic solvents or vehicles include fatty oils, forexample, sesame oil, or synthetic fatty acid esters, (e.g., ethyl oleateor triglycerides). Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol and/or dextran. Optionally, thesuspension may also contain stabilizers. Liposomes and other non-viralvectors can also be used to encapsulate the agent for delivery into thecell.

[0402] The pharmaceutical formulation for systemic administrationaccording to the invention may be formulated for enteral, parenteral, ortopical (top) administration. Indeed, all three types of formulationsmay be used simultaneously to achieve systemic administration of theactive ingredient.

[0403] Suitable formulations for oral administration include hard orsoft gelatin capsules, pills, tablets, including coated tablets,elixirs, suspensions, syrups or inhalations and controlled release formsthereof.

[0404] Potentially, any compound which binds Dkk or a Dkk interactingprotein or modulates the Dkk/LRP5 or Dkk/LRP6 or Dkk/Dkk interactingprotein complex may be a therapeutic compound. In one embodiment of theinvention, a peptide or nucleic acid aptamer according to the inventionis used in a therapeutic composition. Such compositions may comprise anaptamer, or a LRP5 or LRP6 fragment unmodified or modified. In anotherembodiment, the therapeutic compound comprises a Dkk-1 interactingprotein, or biologically active fragment thereof.

[0405] Nucleic acid aptamers have been used in compositions for exampleby chemical bonding to a carrier molecule such as polyethylene glycol(PEG) which may facilitate uptake or stabilize the aptamer. Adi-alkylgylcerol moiety attached to an RNA will embed the aptamer inliposomes, thus stabilizing the compound. Incorporating chemicalsubstitutions (i.e. changing the 2′OH group of ribose to a 2′NH in RNAconfers ribonuclease resistance) and capping, etc. can preventbreakdown. Several such techniques are discussed for RNA aptamers inBrody and Gold (Rev. Mol. Biol. 74:3-13 (2000)).

[0406] Peptide aptamers may by used in therapeutic applications by theintroduction of an expression vector directing aptamer expression intothe affected tissue such as for example by retroviral delivery, byencapsulating the DNA in a delivery complex or simple by naked DNAinjection. Or, the aptamer itself or a synthetic analog may be useddirectly as a drug. Encapsulation in polymers and lipids may assist indelivery. The use of peptide aptamers as therapeutic and diagnosticagents is reviewed by Hoppe-Syler and Butz (J. Mol. Med. 78:426430(2000)).

[0407] In another aspect of the invention. The structure of aconstrained peptide aptamer of the invention may be determined such asby NMR or X-ray crystallography. (Cavanagh et al., Protein NMRSpectroscopy: Principles and Practice, Academic Press, 1996; Drenth,Principles of Protein X-Ray Crystallography, Springer Verlag, 1999)Preferably the structure is determined in complex with the targetprotein. A small molecule analog is then designed according to thepositions of functional elements of the 3D structure of the aptamer.(Guidebook on Molecular Modeling in Drug Design, Cohen, Ed., AcademicPress, 1996; Molecular Modeling and Drug Design (Topics in Molecular andStructural Biology), Vinter and Gardner Eds., CRC Press, 1994) Thus thepresent invention provides a method for the design of effective andspecific drugs which modulate the activity of Dkk, Dkk interactingproteins, Dkk/Dkk interacting protein complex and the Dkk/LRP complex.Small molecule mimetics of the peptide aptamers of the present inventionare encompassed within the scope of the invention.

[0408] In practicing the methods of this invention, the compounds ofthis invention may be used alone or in combination, or in combinationwith other therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be co-administeredalong with other compounds typically prescribed for these conditionsaccording to generally accepted medical practice. For example, thecompounds of this invention can be administered in combination withother therapeutic agents for the treatment of bone loss. Bone lossmediating agents include bone resorption inhibitors such asbisphosphonates (e.g., alendronic acid, clodronic acid, etidronic acid,pamidronic acid, risedronic acid and tiludronic acid), vitamin D andvitamin D analogs, cathepsin K inhibitors, hormonal agents (e.g.,calcitonin and estrogen), and selective estrogen receptor modulators orSERMs (e.g., raloxifene). And bone forming agents such as parathyroidhormone (PTH) and bone morphogenetic proteins (BMP).

[0409] Additionally contemplated are combinations of agents whichregulate Dkk-1 and agents which regulate lipid levels such as HMG-COAreductase inhibitors (i.e., statins such as Mevacor®, Lipitor® and otherinhibitors such as Baycol®, Lescol®, Pravachol® and Zocor®), bile acidsequestrants (e.g., Colestid® and Welchol®), fibric acid derivatives(Atromid-S®, Lopid®, Tricor®), and nicotinic acid. [0001] The compoundsof this invention can be utilized in vivo, ordinarily in vertebrates andpreferably in mammals, such as humans, sheep, horses, cattle, pigs,dogs, cats, rats and mice, or in vitro.

[0410] 14. Transgenic Animals

[0411] Transgenic animal models can be created which conditionallyexpress Dkk and/or LRP5 or LRP6 and/or Dkk interacting proteins, such asthose shown in FIG. 5. These animals can be used as research tools forthe study of the physiological effects of the Dkk-1/Dkk-1 interactingprotein interaction and/or the LRP5 / Dkk interaction. Alternatively,transgenic animals can be created which express a transgenic form of Dkkalone or in addition to a transgenic form of HBM or express Dkkinteracting proteins alone or in addition to a transgenic form of Dkk.Transgenic animals expressing HBM or LRP5 can be crossed with transgenicanimals expressing Dkk or Dkk interacting proteins to obtainheterozygote as well as homozygote animals which express both desiredgenes.

[0412] Animal models may be created to directly modulate the Dkk/Dkkinteracting protein or Dkk/LRP5 interaction activity in vivo to serve asa research tool for determining the efficacy of candidate compoundswhich modulate the Dkk/Dkk interacting protein or LRP5/Dkk interactionactivity in vitro. Animals, such as transgenic mice, can be createdusing the techniques employed to make transgenic mice that express forexample, human Dkk or a Dkk interacting protein, or knockouts (KO),which may be conditional, of the gene encoding mouse Dkk or Dkkinteracting protein. Knock-in animals include animals wherein genes havebeen introduced and animals wherein a gene that was previouslyknocked-out is reintroduced into the animal. Other transgenic animalscan be created with inducible forms of Dkk or a Dkk interacting proteinto study the effects of the gene on bone mass development and loss aswell as lipid level regulation. These animals can also be used to studylong term effects of Dkk or Dkk interacting protein modulation.Transgenic animals may be created to express peptide aptamers, orproduce RNA aptamers. The transgenic vectors may direct expression in atissue specific manner by the use of tissue specific promoters. In apreferred embodiment, a peptide aptamer fusion protein is expressedusing a bone specific promoter. Such systems can provide a tissuespecific knock-out of Dkk or Dkk interacting protein activity.

[0413] General methods for creating transgenic animals are known in theart, and are described in, for example, Strategies in Transgenic AnimalScience (Glenn M. Monastersky and James M. Robl eds., ASM Press;Washington, D.C., 1995); Transgenic Animal Technology: A LaboratoryHandbook (Carl A. Pinkert ed., Academic Press 1994); Transgenic Animals(Louis Marie Houdebine, ed., Harwood Academic Press, 1997);Overexpression and Knockout of Cytokines in Transgenic Mice (Chaim O.Jacob, ed., Academic Press 1994); Microinjection and Transgenesis:Strategies and Protocols (Springer Lab Manual) (Angel Cid-Arregui andAlejandro Garcia-Carranca, eds., Springer Verlag 1998); and Manipulatingthe Mouse Embryo: A Laboratory Manual (Brigid Hogan et al., eds., ColdSpring Harbor Laboratory Press 1994).

[0414] 15. Peptide and Nucleotide Aptamers and Peptide Aptamer Mimetics

[0415] Another embodiment contemplates the use of peptide and nucleotideaptamer technology to screen for agents which interact with Dkk, whichblock Dkk from interacting with LRP5 or LRP6, or which block any otherDkk ligand interaction, or which interact with Dkk interacting proteins,such as those shown in FIG. 5. Peptide aptamers are molecules in which avariable peptide domain is displayed from a scaffold protein.Thioredoxin A (trxA) is commonly used for a scaffold. The peptide insertdestroys the catalytic site of trxA. It is recognized that numerousproteins may also be used as scaffolding proteins to constrain and/orpresent a peptide aptamer. Other scaffold proteins that could display aconstrained peptide aptamer could include staphylococcal nuclease, theprotease inhibitor eglin C, the Streptomyces tendea alpha-amylaseinhibitor Tendamistat, Sp1, and green fluorescent protein (GFP)(reviewed in Hoppe-Seyler et al., J. Steroid Biochem Mol. Biol.78:105-11 (2001)), and the S1 nuclease from Staphylococcus or M13 forphage display. Any molecule to which the aptamer could be anchored andpresented in its bioactive conformation would be suitable.

[0416] Aptamers can then specifically bind to a given target protein invitro and in vivo and have the potential to selectively block thefunction of their target protein. Peptide aptamers are selected fromrandomized expression libraries on the basis of their in vivo bindingcapacity to the desired target protein. Briefly, a target protein (e.g.,Dkk, a Dkk interacting protein, or LRP5/6) is linked to a heterologousDNA binding domain (BD) and expressed as bait in a yeast test strain.Concomitantly, a library coding for different peptides (e.g., 16-mers)of randomized sequence inserted in a scaffold protein sequence, whichare linked to a heterologous transcriptional activation domain (AD) isexpressed as prey. If a peptide binds to a target protein, a functionaltranscription factor is reconstituted, in which the BD and AD arebridged together by interacting proteins. This transcription factor isthen able to activate the promoter of a marker gene which can bemonitored by colorimetric enzymatic assays or by growth selection.Additional variation, methods of preparing and screening methodologiesare described in, for example, Hoppe-Seyler et al., J. Mol Med. 78:426-430 (2000). Nucleotide aptamers are described for example in Brodyet al., Trends Mol. Biotechnol. 74: 5-13 (2000). Additional methods ofmaking and using nucleotide aptamers include SELEX, i.e., SystematicEvolution of Ligands by Exponential Enrichment. SELEX is a process ofisolating oligonucleotide ligands of a chosen target molecule (see Tuerkand Gold, Science 249:505-510 (1990); U.S. Pat. Nos. 5,475,096,5,595,877, and 5,660,985). SELEX, as described in Tuerk and Gold,involves admixing the target molecule with a pool of oligonucleotides(e.g., RNA) of diverse sequences; retaining complexes formed between thetarget and oligonucleotides; recovering the oligonucleotides bound tothe target; reverse-transcribing the RNA into DNA; amplifying the DNAwith polymerase chain reactions (PCR); transcribing the amplified DNAinto RNA; and repeating the cycle with ever increasing bindingstringency. Three enzymatic reactions are required for each cycle. Itusually takes 12-15 cycles to isolate aptamers of high affinity andspecificity to the target. An aptamer is an oligonucleotide that iscapable of binding to an intended target substance but not othermolecules under the same conditions.

[0417] In another reference, Bock et al., Nature 355:564-566 (1990),describe a different process from the SELEX method of Tuerk and Gold inthat only one enzymatic reaction is required for each cycle (i.e., PCR)because the nucleic acid library in Bock's method is comprised of DNAinstead of RNA. The identification and isolation of aptamers of highspecificity and affinity with the method of Bock et al. still requiresrepeated cycles in a chromatographic column.

[0418] Other nucleotide aptamer methods include those described byConrad et al., Meth. Enzymol. 267:336-367 (1996). Conrad et al. describea variety of methods for isolating aptamers, all of which employrepeated cycles to enrich target-bound ligands and require a largeamount of purified target molecules. More recently described methods ofmaking and using nucleotide aptamers include, but are not limited tothose described in U.S. Pat. Nos. 6,180,348; 6,051,388; 5,840,867;5,780,610, 5,756,291 and 5,582,981.

[0419] Potentially, any compound which binds Dkk or a Dkk interactingprotein or modulates the Dkk/Dkk interacting protein or Dkk/LRP5 orDkk/LRP6 complex may be a therapeutic compound. In one embodiment of theinvention, a peptide or nucleic acid aptamer according to the inventionis used in a therapeutic composition. Such compositions may comprise anaptamer, or a LRP5 or LRP6 fragment unmodified or modified.

[0420] Nucleic acid aptamers have been used in compositions for exampleby chemical bonding to a carrier molecule such as polyethylene glycol(PEG) which may facilitate uptake or stabilize the aptamer. Adi-alkylglycerol moiety attached to an RNA will embed the aptamer inliposomes, thus stabilizing the compound. Incorporating chemicalsubstitutions (i.e., changing the 2′-OH group of ribose to a 2′-NH inRNA confers ribonuclease resistance) and capping, etc. can preventbreakdown. Several such techniques are discussed for RNA aptamers inBrody and Gold Rev. Mol. Biol. 74:3-13 (2000).

[0421] Peptide aptamers may by used in therapeutic applications by theintroduction of an expression vector directing aptamer expression intothe affected tissue such as for example by retroviral delivery, byencapsulating the DNA in a delivery complex or simple by naked DNAinjection. Or, the aptamer itself or a synthetic analog may be useddirectly as a drug. Encapsulation in polymers and lipids may assist indelivery. The use of peptide aptamers as therapeutic and diagnosticagents is reviewed by Hoppe-Syler and Butz J. Mol. Med. 78:426-430(2000).

[0422] In another aspect of the invention, the structure of aconstrained peptide aptamer of the invention may be determined such asby NMR or X-ray crystallography. (Cavanagh et al., Protein NMRSpectroscopy: Principles and Practice, Academic Press, 1996; Drenth,Principles of Protein X-Ray Crystallography, Springer Verlag, 1999)Preferably the structure is determined in complex with the targetprotein. A small molecule analog is then designed according to thepositions of functional elements of the 3D structure of the aptamer.(Guidebook on Molecular Modeling in Drug Design, Cohen, Ed., AcademicPress, 1996; Molecular Modeling and Drug Design (Topics in Molecular andStructural Biology), Vinter and Gardner Eds., CRC Press, 1994) Thus, amethod is provided for the design of effective and specific drugs whichmodulate the activity of Dkk, Dkk interacting proteins, Dkk/Dkkinteracting protein complex, and the Dkk/LRP complex. Small moleculemimics of the peptide aptamers of the present invention are alsoencompassed within the scope of the invention.

[0423] 16. Alternative Variants of LRP5/LRP6 Having HBM Activity

[0424] A structural model of the LRP5/Zmax1 first beta-propeller modulewas generated based on a model prediction in Springer et al., (1998) J.Molecular Biology, 283:837-862. Based on the model, certain amino acidresidues were identified as important variants of LRP5/HBM/Zmax1. Thefollowing three categories provide examples of such variants:

[0425] The shape of the beta-propeller resembles a disk withinward-sloping sides and a hole down the middle. Residue 171 is in aloop on the outer or top surface of the domain in blade 4 of propellermodule 1. Thus, variants comprising changed residues in structurallyequivalent positions in other blades; as well as residues that areslightly more interior to the binding pocket, but still accessible tothe surface, are important embodiments of the present invention for thestudy of bone mass modulation by LRP5/HBM, for the development ofpharmaceuticals and treatments of bone mass disorders, and for otherobjectives of the present invention. The following are examples of suchvariants:

[0426] A214V ( a position equivalent to 171 in blade 5; alanine is notconserved in other propellers),

[0427] E128V (a position equivalent to 171 in blade 3; glutamate is notconserved in other propellers),

[0428] A65V (a position equivalent to 171 in blade 2; alanine isconserved in propellers 1-3 but not 4),

[0429] G199V (an accessible interior position in blade 5; glycine isconserved in propellers 1-3 but not 4), and

[0430] M282V (accessible interior position in blade 1; methionine isconserved in propellers 1-3 but not 4).

[0431] LRP5/Zmax1 has four beta-propeller structures; the first threebeta-propeller modules conserve a glycine in the position correspondingto residue 171 in human LRP5/Zmax1. Therefore, variants bearing a valinein the equivalent positions in the other propellers are importantembodiments of the present invention. The following variants are usefulfor the study of bone mass modulation by LRP5/HBM, for the developmentof pharmaceuticals and treatments of bone mass disorders, and for otherobjectives of the present invention: G479V, G781V, and Q1087V.

[0432] The G171V HBM polymorphism results in “occupied space” of thebeta-propeller 1, with the side-chain from the valine residue stickingout into an open binding pocket and potentially altering aligand/protein interaction. The glycine residue is conserved inLRP5/Zmax1 propellers 1, 2 and 3 but is a glutamine in propeller 4.Therefore, the following variants of LRP5/HBM are important embodimentsof the present invention for the study of bone mass modulation byLRP5/HBM, for the development of pharmaceuticals and treatments of bonemass disorders, and for other objectives of the present invention:

[0433] G171K (which introduces a charged side-chain),

[0434] G171F (which introduces a ringed side-chain),

[0435] G171I (which introduces a branched side-chain), and

[0436] G171Q (which introduces the propeller 4 residue).

[0437] Furthermore, LRP6 is the closest homolog of LRP5/Zmax1. LRP6 hasa beta-propeller structure predicted to be similar, if not identical toZmax1. The position corresponding to glycine 171 in human LRP5/Zmax1 isglycine 158 of human LRP6. Thus, corresponding variants of LRP6 are animportant embodiment of the present invention for the study of thespecificity of LRP5/Zmax1 versus its related family member, for thedevelopment of pharmaceuticals and treatments of bone mass disorders,and for other objectives of the present invention. Specifically, forexample, a glycine to valine substitution at the structurally equivalentposition, residue 158, of human LRP6 and similar variants of otherspecies' LRP6 homologs represent important research tools.

[0438] Site-directed mutants of LRP5 were generated in the full-lengthhuman LRP5 cDNA using the QuikChange XL-Site-Directed Mutagenesis Kit(catalog #200516, Stratagene, La Jolla, Calif.) following themanufacturer's protocol. The mutant sequences were introduced usingcomplementary synthetic oligonucleotides: A65V:TGGTCAGCGGCCTGGAGGATGTGGCCGCAGTGGACTTCC (SEQ ID NO:129) andGGAAGTCCACTGCGGCCACATCCTCCAGGCCGCTGACCA (SEQ ID NO:130) E128V:AAGCTGTACTGGACGGACTCAGTGACCAACCGCATCGAGG (SEQ ID NO:131) andCCTCGATGCGGTTGGTCACTGAGTCCGTCCAGTACAGCTT (SEQ ID NO:132) G171K:ATGTACTGGACAGACTGGAAGGAGACGCCCCGGATTGAGCG (SEQ ID NO:133) andCGCTCAATCCGGGGCGTCTCCTTCCAGTCTGTCCAGTACAT (SEQ ID NO:134) G171F:ATGTACTGGACAGACTGGTTTGAGACGCCCCGGATTGAGCG (SEQ ID NO:135) andCGCTCAATCCGGGGCGTCTCAAACCAGTCTGTCCAGTACAT (SEQ ID NO:136) G171I:ATGTACTGGACAGACTGGATTGAGACGCCCCGGATTGAGCG (SEQ ID NO:137) andCGCTCAATCCGGGGCGTCTCAATCCAGTCTGTCCAGTACAT (SEQ ID NO:138) G171Q:ATGTACTGGACAGACTGGCAGGAGACGCCCCGGAUGAGCG (SEQ ID NO:139) andCGCTCAATCCGGGGCGTCTCCTGCCAGTCTGTCCAGTACAT (SEQ ID NO:140) G199V:CGGACATTTACTGGCCCAATGTACTGACCATCGACCTGGAGG (SEQ ID NO:141) andCCTCCAGGTCGATGGTCAGTACATTGGGCCAGTAAATGTCCG (SEQ ID NO:142) A214V:AGCTCTACTGGGCTGACGTCAAGCTCAGCTTCATCCACCG (SEQ ID NO:143) andCGGTGGATGAAGCTGAGCTTGACGTCAGCCCAGTAGAGCT (SEQ ID NO:144) M282V:GAGTGCCCTCTACTCACCCGTGGACATCCAGGTGCTGAGCC (SEQ ID NO:145) andGGCTCAGCACCTGGATGTCCACGGGTGAGTAGAGGGCACTC (SEQ ID NO:146) G479V:CATGTACTGGACAGACTGGGTAGAGAACCCTAAAATCGAGTGTGC (SEQ ID NO:147) andGCACACTGGATTTTAGGGTTCTCTACCCAGTCTGTCCAGTACATG (SEQ ID NO:148) G781V:CATCTACTGGACCGAGTGGGTCGGCAAGCCGAGGATCGTGCG (SEQ ID NO:149) andCGCACGATCCTCGGCTTGCCGACCCACTCGGTCCAGTAGATG (SEQ ID NO:150) Q1087V:GTACTTCACCAACATGGTGGACCGGGCAGCCAAGATCGAACG (SEQ ID NO:151) andCGTTCGATCTTGGCTGCCCGGTCCACCATG1TGGTGAAGTAC (SEQ ID NO:152) LRP6 G158V:GTACTGGACAGACTGGGTAGAAGTGCCIMAGATAGAACGTGC (SEQ ID NO:153) andGCACGTTCTATCTTTGGCACTTCTACCCAGTCTGTCCAGTAC. (SEQ ID NO:154)

[0439] All constructs were sequence verified to ensure that only theengineered modification was present in the gene. Once verified, eachvariant was functionally evaluated in the TCF-luciferase assay in U2OScells (essentially as described in Example 7. Other functionalevaluations could also be performed, such as the Xenopus embryo assay(essentially as described in Example 6), or other assays to evaluate Wntsignaling, Dkk modulation, or anabolic bone effect. Binding of thesemutants to Dkk, LRP-interacting proteins, Dkk-interacting proteins, orpeptide aptamers to any of the preceding could also be investigated in avariety of ways such as in a two-hybrid system (such as in yeast asdescribed in this application), or other methods.

[0440]FIG. 24 shows the effects of the G171F mutation in propeller 1 ofLRP5. This mutation is at the same position as HBM's G171V substitution.Expression of G171F results in an HBM effect. That is, in the presenceof Wnt, G171F is able to activate the TCF-luciferase reporter construct.In fact, it may activate the reporter to a greater extent than eitherLRP5 or HBM. Furthermore, in the presence of Dkk1 and Wnt1, G171F isless susceptible than LRP5 to modulation by Dkk. These data exemplifythat the G171F variant modulates Wnt signaling in a manner similar toHBM. In addition, this data confirms that HBM's valine residue at 171 isnot the only modification at 171 that can result in an HBM effect.Together these data support an important role for LRP5 propeller 1 inmodulating Wnt pathway activity; in responding to Dkk modulation; and,in the ability to generate an HBM effect.

[0441]FIG. 25 shows the effects of the M282V mutation in propeller 1 ofLRP5. M282 expression results in an HBM-effect. That is, in the presenceof Wnt, M282 is able to activate the TCF-luciferase reporter construct.Furthermore, in the presence of Dkk1 and Wnt1, M282V is less susceptiblethan LRP5 to modulation by Dkk. These data show that the M282V variantmodulates Wnt signaling in a manner similar to HBM. In addition, thisdata confirms that modifications of other residues in propeller 1 ofLRP5 can result in an HBM effect.

[0442] These data support an “occupied space” model of the HBM mutationin propeller 1 and show that multiple mutations of propeller 1 arecapable of generating an HBM effect; the original G171V HBM mutation isnot unique in this ability. Moreover, various perturbations in propeller1 can modulate Dkk activity.

[0443] These data illustrate the molecular mechanism of Dkk modulationof LRP signaling. Using the methods disclosed herein and in U.S.Application 60/290,071, generation of a comprehensive mutant panel willreveal residues in LRP that function in Dkk modulation of Wnt signaling.Such variants of LRP5 and LRP6 that modulate Dkk activity and theresidues which distinguish them from LRP5 and LRP6 are points fortherapeutic intervention by small molecule compound, antibody, peptideaptamer, or other agents. Furthermore, models of each HBM-effectmutation/polymorphism may be used in rational drug design of an HBMmimetic agent.

[0444] These are only a few illustrative examples presented to betterdescribe the present invention. Variants of LRP5 which have demonstratedHBM activity in assays include G171F, M282V, G171K, G171Q and A214V.Clearly, other variants may be contemplated within the scope of thepresent invention. Furthermore, wherever HBM is recited in the methodsof the invention, it should be understood that any such alternativevariant of LRP which demonstrates HBM biological activity is alsoencompassed by those claims.

[0445] 17. Screening Assays

[0446] The two-hybrid system is extremely useful for studyingprotein:protein interactions. See, e.g., Chien et al., Proc. Natl Acad.Sci. USA 88:9578-82 (1991); Fields et al., Trends Genetics 10:286-92(1994); Harper et al., Cell 75:805-16 (1993); Vojtek et al., Cell74:205-14 (1993); Luban et al., Cell 73:1067-78 (1993); Li et al., FASEBJ. 7:957-63 (1993); Zang et al., Nature 364:308-13 (1993); Golemis etal., Mol. Cell. Biol. 12:3006-14 (1992); Sato et al., Proc. Natl Acad.Sci. USA 91:9238-42 (1994); Coghlan et al.; Science 267:108-111 (1995);Kalpana et al., Science 266:2002-6 (1994); Helps et al., FEBS Lett.340:93-8 (1994); Yeung et al., Genes & Devel. 8:2087-9 (1994); Durfee etal., Genes & Devel. 7:555-569 (1993); Paetkau et al., Genes & Devel.8:2035-45; Spaargaren et al., 1994 Proc. Natl. Acad. Sci. USA91:12609-13 (1994); Ye et al., Proc. Natl Acad. Sci. USA 91:12629-33(1994); and U.S. Pat. Nos. 5,989,808; 6,251,602; and 6,284,519.

[0447] Variations of the system are available for screening yeastphagemid (see, e.g., Harper, Cellular Interactions and Development: APractical Approach, 153-179 (1993); Elledge et al., Proc. Natl Acad.Sci. USA 88:1731-5 (1991)) or plasmid (Bartel, 1993 and Bartel, Cell14:920-4 (1993)); Finley et al., Proc. Natl Acad. Sci. USA 91:12980-4(1994)) cDNA libraries to clone interacting proteins, as well as forstudying known protein pairs.

[0448] The success of the two-hybrid system relies upon the fact thatthe DNA binding and polymerase activation domains of many transcriptionfactors, such as GAL4, can be separated and then rejoined to restorefunctionality (Morin et al., Nuc. Acids Res. 21:2157-63 (1993)). Whilethese examples describe two-hybrid screens in the yeast system, it isunderstood that a two-hybrid screen may be conducted in other systemssuch as mammalian cell lines. The invention is therefore not limited tothe use of a yeast two-hybrid system, but encompasses such alternativesystems.

[0449] Yeast strains with integrated copies of various reporter genecassettes, such as for example GAL.fwdarw.LacZ, GAL.fwdarw.HIS3 orGAL.fwdarw.URA3 (Bartel, in Cellular Interactions and Development: APractical Approach, 153-179 (1993); Harper et al., Cell 75:805-16(1993); Fields et al., Trends Genetics 10:286-92 (1994)) areco-transformed with two plasmids, each expressing a different fusionprotein. One plasmid encodes a fusion between protein “X” and the DNAbinding domain of, for example, the GAL4 yeast transcription activator(Brent et al., Cell 43:729-36 (1985); Ma et al., Cell 48:847-53 (1987);Keegan et al., Science 231:699-704 (1986)), while the other plasmidencodes a fusion between protein “Y” and the RNA polymerase activationdomain of GAL4 (Keegan et al., 1986). The plasmids are transformed intoa strain of the yeast that contains a reporter gene, such as lacZ, whoseregulatory region contains GAL4 binding sites. If proteins X and Yinteract, they reconstitute a functional GAL4 transcription activatorprotein by bringing the two GAL4 components into sufficient proximity toactivate transcription. It is well understood that the role of bait andprey proteins may be alternatively switched and thus the embodiments ofthis invention contemplate and encompass both alternative arrangements.

[0450] Either hybrid protein alone must be unable to activatetranscription of the reporter gene, the DNA-binding domain hybrid,because it does not provide an activation function, and the activationdomain hybrid, because it cannot localize to the GAL4 binding sites.Interaction of the two test proteins reconstitutes the function of GAL4and results in expression of the reporter gene. The reporter genecassettes consist of minimal promoters that contain the GAL4 DNArecognition site (Johnson et al., Mol. Cell. Biol. 4:1440-8 (1984);Lorch et a., J. Mol. Biol. 186:821-824 (1984)) cloned 5′ to their TATAbox. Transcription activation is scored by measuring either theexpression of β-galactosidase or the growth of the transformants onminimal medium lacking the specific nutrient that permits auxotrophicselection for the transcription product, e.g., URA3 (uracil selection)or HIS3 (histidine selection). See, e.g., Bartel, 1993; Durfee et al.,Genes & Devel. 7:555-569 (1993); Fields et al., Trends Genet. 10:286-292(1994); and U.S. Pat. No. 5,283,173.

[0451] Generally, these methods include two proteins to be tested forinteraction which are expressed as hybrids in the nucleus of a yeastcell. One of the proteins is fused to the DNA-binding domain (DBD) of atranscription factor and the other is fused to a transcriptionactivation domain (AD). If the proteins interact, they reconstitute afunctional transcription factor that activates one or more reportergenes that contain binding sites for the DBD. Exemplary two-hybridassays which have been used for Dkk-1 or Dkk-1/LRP5 are presented in theExamples below.

[0452] Additional methods of preparing two hybrid assay systems forDkk-1 interactors would be evident to one of ordinary skill in the art.See for example, Finley et al., “Two-Hybrid Analysis of GeneticRegulatory Networks,” in The Yeast Two-Hybrid System (Paul L. Bartel etal., eds., Oxford, 1997); Meijia Yang, “Use of a Combinatorial PeptideLibrary in the Two-Hybrid Assay,” in The Yeast Two-Hybrid System (PaulL. Bartel et al., eds., Oxford, 1997); Gietz et a., “Identification ofproteins that interact with a protein of interest: Applications of theyeast two-hybrid system,” Mol. & Cell. Biochem. 172:67-9 (1997); K. H.Young, “Yeast Two-Hybrid: So Many Interactions,(in) so Little Time,”Biol. Reprod. 58:302-311 (1998); R. Brent et al., “Understanding Geneand Allele Function with Two-Hybrid Methods,” Annu. Rev. Genet31:663-704 (1997). It will be appreciated that protein networks can beelucidated by performing sequential screens of activation domain-fusionlibraries.

[0453] Without further description, it is believed that one of ordinaryskill in the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure.

EXAMPLES

[0454] The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well-known inthe art or the techniques specifically described below were utilized.

[0455] For routine practice of the protocols referenced below, one ofskill in the art is directed to the references cited in this applicationas well as the several Current Protocol guides, which are continuouslyupdated, widely available and published by John Wiley and Sons, (NewYork). In the life sciences, Current Protocols publishes comprehensivemanuals in Molecular Biology, Immunology, Human Genetics, ProteinScience, Cytometry, Neuroscience, Pharmacology, Cell Biology,Toxicology, and Nucleic Acid Chemistry. Additional sources are known toone of skill in the art.

Example 1 Yeast Two Hybrid Screen Using LRP5 Ligand Binding Domain (LBD)Bait Sequences

[0456] In a screen against human osteoblast library (i.e., HOB03C5, acustom Gibco generated Y2H compatible cDNA library from a humanosteoblast cell line as described by Bodine and Komm, Bone 25:535-43(1999)), an interaction with Dkk-1 was identified. The LRP5 ligandbinding domain (LBD) baits used for this screen are depicted in FIGS. 2Band C. The basic protocol is as follows:

[0457] An overnight culture of the yeast strain containing the bait ofinterest is grown in 20 ml of appropriate selective medium containing 2%glucose at 30° C. The overnight culture is diluted by a 10 fold factorinto YPDmedia supplemented with 40 mg/l of adenine, and grown for 4hours at 30° C.

[0458] For each mating event, an aliquot of the frozen prey library isgrown in 150 ml YAPD medium for 5 hours at 30° C.

[0459] Appropriate volumes calculated by measuring the OD600 of eachculture are combined into a tube. The number of diploids to be screenedis typically ten times the number of clones originally present in theprey library of interest. Assuming a mating efficiency of 20% minimum,fifty times (i.e., ten times coverage multiplied by 20% matingefficiency) as many haploid cells containing the bait and as many cellscontaining the prey are used in any given mating event. The mixture isfiltered over a 47 mm, 0.45 mm sterile Metricel filter membrane(Gelman).

[0460] Using sterile forceps, the filter is transferred onto a 100 mm²YAPD agar plate with the cell side up, removing all air bubblesunderneath the filter. The plate is incubated overnight at roomtemperature.

[0461] The filter is transferred into a 50 ml Falcon tube using sterileforceps and 10 ml SD medium containing 2% glucose are added to resuspendthe cells. The filter, once free of cells, is removed and the cellsuspension is spun for 5 min. at 2,000 xg.

[0462] The cells are resuspended in 10 ml SD medium containing 2%glucose. An aliquot of 100 μl is set aside for titration.

[0463] The cells are plated onto large square plates containingappropriate selective media and incubated at 30° C. for three to fivedays.

[0464] To calculate the mating efficiency and to determine the totalnumber of diploid cells screened, the 100 μl aliquot set aside fortitration is diluted and plated onto different selective media. Themating efficiency is calculated by dividing the number of diploids/ml bythe lowest number of haploids/ml, either bait or prey, and multiplied by100. For example, if 2 million diploids were obtained by mating 10million of haploids containing a bait and 12 million of haploidscontaining a prey, then the mating efficiency is calculated by dividing2 million by 10 million, which equals 0.2 and multiplied by 100 whichequals 20%. Typical mating efficiencies under the above conditions arewithin about 20 to about 40%. The total number of diploids screened in amating event is obtained by multiplying the number of diploids/ml by thetotal number of ml plated, typically about 10.

[0465] Isolation of Colonies Containing Pairs of Interacting Proteins

[0466] Yeast colonies from the interaction selection (large square)plates are picked with a sterile toothpick and patched onto platescontaining the appropriate selective media and incubated at 30° C. fortwo days.

[0467] To further ensure purity of the yeast, the plates are replicatedonto another plate containing the same media and incubated at 30° C. foranother two days.

[0468] Yeast patches are scraped using a sterile toothpick and placedinto a 96-well format plate containing 100 μl SD-L-W-H with 2% glucoseliquid medium.

[0469] Half the volume of the plate is transferred to a 96-well platecontaining 50 μl of 40% glycerol for storage. The other half is setaside for replication and galactosidase-activity assay (see below).

[0470] Cells are replicated onto a SD-L-W-H plate with 2% glucose plateto create a master plate, and incubated two days at 30° C. The masterplate is replicated onto different selective media to score the strengthof each interaction.

[0471] Cells are also replicated onto media selecting for the preyvector only for colony PCR and incubated two days at 30° C.

[0472] Galactosidase Activity Assay

[0473] Ten microliters from the 96-well plate (set aside from above) aretransferred into another 96-well plate containing 100 μl SD and 2%glucose media. The cell density is measured at OD₆₀₀ using aspectrophotometer, the OD₆₀₀ is usually between 0.03 and 0.1. Fiftymicroliters of Galactosidase reaction mixture (Tropix) are added tomicroplates (Marsh) specifically designed for the luminometer (HewlettPackard Lumicount). Fifty microliters of the diluted cells are thenadded and mixed by pipetting. The reaction is incubated sixty to onehundred twenty minutes at room temperature. Relative Light Units (RLUs)are read by the luminometer. Each plate contains a negative control,constituted by diploid yeast containing the bait of interest and anempty prey vector. To be scored as positive, the diploids tested have tohave an RLU number at least twice as high as the negative control.

Example 2 Minimum Interaction Domain Mapping

[0474] Further analysis of yeast two hybrid (Y2H) interacting proteinsincludes the dissection of protein motifs responsible for theinteraction. Sequence alignment of multiple clones identified in the Y2Hscreens can help identify the smallest common region responsible for theinteraction. In the absence of appropriate clones, deletion mapping ofinteracting domains is necessary.

[0475] PCR primers containing restriction sites suitable for cloning aredesigned to cover multiple sub-domains of the protein of interest (baitor prey). The methods involved in cloning, sequencing, yeasttransformation, mating, and scoring of interactions are readilyperformed by one of ordinary skill in the art of molecular biology andgenetic engineering.

[0476] Materials and Methods

[0477] Minimum interaction domain: primers were designed for PCR of theDkk-1 clone isolated by screening a primary osteoblast cell strain(HOB03C5) library with pooled Zmax1/LRP5 ligand binding domain (LBD)baits: LBD1 (Leu969-Pro1376) and LBD4 (Arg1070-Pro1376). The primers,which are presented in 5′ to 3′ orientation, were as follows: SEQ ID NOPrimer Sequence 155 Forward 1 TTTTTTGTCGACCAATTCCAACGCTATCAAG 156Forward 2 TTTTTTGTCGACCTGCGCTAGTCCCACCCGC 157 Forward 3TTTTTTGTCGACCGTGTCTTCTGATCAAAATC 158 Forward 4TTTTTTGTCGACCGGACAAGAAGGTTCTGTTTG 159 Reverse 1TTTTTTGCGGCCGCTTATTTGGTGTGATACATTTTTG 160 Reverse 2TTTTTTGCGGCCGCTTAGCAAGACAGACCTTCTCC 161 Reverse 3TTTTTTGCGGCCGCTTAGTGTCTCTGACAAGTGTG

[0478] PCR was performed using PfuTurbo® polymerase (Stratagene). ThePCR products were gel purified, digested with SalI/NotI and ligated topPC86 (Gibco/BRL) which had been linearized with SalI/NotI. Clones wererecovered and sequenced to ascertain that the structure was as expectedand that the Gal4 activation domain and Dkk-1 were in-frame. The ORF ofDkk-1 was Metl-His266, as in human Dkk-1 (GenBank Accession No.XM_(—)005730).

[0479] The clones used were as follows: D5 (F1/R3: Asn34-His266), D4(F1/R2: Asn34-Cys245), D3 (F1/R1: Asn34-Lys182), D9 (F2/R3:Cys97-His266), D12 (F3/R3, val 139-His266), D14 (F4/R3: Gly183-His266),D8 (F2/R2: Cys97-Cys245), and D11 (F3/R2: Val39-Cys245). F1, F2, F3 andF4 refer respectively to Forward primers 1, 2, 3 and 4. R1, R2 and R3refer respectively to reverse primers 1, 2 and 3.

[0480] These clones were transformed into yeast and mated with each ofthree yeast strains containing pDBleu (Gibco/BRL), pDBleuLBD1, andpDBleuLBD4. Positive interactions were detected by growth of the hybridson appropriate selective media.

[0481] Results

[0482] Minimum interaction domain: FIG. 6 shows that while growth wasobserved in diploids of D4, D5, D8, D9, and D12, no growth was observedin hybrids of D3, D11, and D12. Carboxy terminal (C-terminal) deletionsindicated that while the C-terminal amino acids of Dkk-1 containing thepotential N-glycosylation site (Arg246-His266) are not required forinteraction with Zmax1/LRP5 LBD baits, the Cys2 domain, Gly183-Cys245,is required. N-terminal deletions also demonstrated that the regionbetween the two cysteine domains, i.e. Val139 to Lys182, is alsorequired. Two minimum interaction domain constructs were isolated: D12(Val139-His266) and D8 (Cys97-Cys245). Similar constructs could beprepared for Dkk-1 interactors.

Example 3 Yeast-2 Hybrid Screen for Peptide Aptamer Sequences to Dkk-1Peptide Aptamer Library Construction

[0483] A peptide aptamer library, Tpep, was constructed, which providesa means to identify chimeric proteins that bind to a protein target (orbait) of interest using classic yeast two hybrid (Y2H) assays. The Tpeplibrary is a combinatorial aptamer library composed of constrainedrandom peptides, expressed within the context of the disulfide loop ofE. coli thioredoxin (trxA), and as C-termini fusion to the S. cerevisiaeGal4 activation domain. The Tpep library was generated using arestriction enzyme modified recombinant Y2H prey vector, pPC86 (Gibco),which contains the trxA scaffold protein.

[0484] Generation of Aptamer-Encoding Sequences

[0485] Aptamer-encoding sequences were produced as follows. DNA encodingrandom stretches of approximately sixteen amino acids surrounded byappropriate restriction sites were generated by semi-randomoligonucleotide synthesis. The synthetic oligonucleotides werePCR-amplified, restriction digested, and cloned into the permissivesites within the trxA scaffold protein. The cloning strategy was toinsert the random oligonucleotide sequence is in-frame with the scaffoldprotein coding sequence, resulting in expression of a scaffoldprotein-aptamer chimera. The scaffold protein is itself in-frame withthe activation domain of Gal4, within the pPC86 vector that isappropriate for the aptamer to be expressed and functional in a regularY2H assay. Additional methods of preparing aptamers would be apparent tothe skilled artisan.

[0486] Generation of a Permissive Recombinant pPC86 Vector Containingthe trxA Coding Sequence

[0487] First the RsrII restriction site located within the Gal4activation domain of pPC86 (Gibco) was eliminated by site-directedmutagenesis (Quickchange™ kit, Stratagene). The amino acid sequence ofthe Gal4 activation domain was unchanged by this modification. Thestrength of different control interactions was verified to be unchangedby the modification.

[0488] Second, the E. coli trxA coding sequence was cloned into the SalIand NotI sites of the RstII-modified pPC86. EcoRI and SpeI sites werethen introduced within the trxA RsrII site. The oligonucleotidesencoding the peptide aptamers were cloned into the EcoRI and SpeI sitesof the resulting vector.

Example 4 Yeast-2 Hybrid Screen for Dkk-1 Interacting Proteins

[0489] A Dkk-1 bait sequence was utilized in a yeast two hybrid screento identify Dkk-1 interacting proteins. The procedure for the Y2H wascarried out similarly to that employed in Example 1, except that theDkk-1 bait from FIG. 2C was used instead of LRP baits. The screen wasperformed using Hela and fetal brain libraries (Invitrogen Corporation,Carlsbad, Calif.). Multiple libraries were used to identify additionalDkk-1 interacting proteins and to confirm interactions found in otherlibraries.

[0490] The list of Dkk-1 interacting proteins uncovered in these Y2Hscreens are listed in FIG. 5.

[0491] The interacting proteins identified in the Dkk-1 bait screen canbe used in other Y2H screens with LRP baits and other Dkk-1 interactingproteins to determine more complex interactions which may modulateDkk-1/LRP interactions and/or Wnt signaling.

Example 5 Generation of Antibodies

[0492] In each of the following antibody-generating examples, thesynthesis of these linear peptides is followed by injection into two NewZealand Rabbits. Subsequent boosts and bleeds are taken according to astandard ten-week protocol. The end-user receives back 5 mgs of peptide,aliquots of pre-bleeds, roughly 80 ml of crude sera from each of the tworabbits and, and ELISA titration data is obtained.

[0493] Generation of LRP5 Polymorphism-Specific Antibodies

[0494] Antibodies were generated to the following peptides to obtainantibodies which distinguish the HBM polymorphism versus wild-typeLRP5/Zmax: MYWTDWVETPRIE (SEQ ID NO:123) (mutant peptide) andMYWTDWGETPRIE (SEQ ID NO:124) (wild-type peptide for negativeselection). Immunofluorescence data confirmed that the antibody, afteraffinity purification, is specific for HBM and does not recognize LRP5(FIG. 17).

[0495] Generation of LRP5 Monospecific Antibodies

[0496] LRP5 monospecific polyclonal antibodies were generated to thefollowing amino acid sequences of LRP5: Peptide 1 (a.a.265-277)—KRTGGKRKEILSA (SEQ ID NO:125), Peptide 2 (a.a.1178-1194)—ERVEKTTGDKRTRIQGR (SEQ ID NO:126), and Peptide 3 (a.a.1352-1375)—KQQCDSFPDCIDGSDE (SEQ ID NO:127). Immunofluorescenceconfirmed that the antibody generated detects LRP5.

[0497] Generation of Dkk-1 Monospecific Polyclonal Antibodies

[0498] Dkk-1 monospecific polyclonal antibodies were generated to thefollowing amino acid sequences of Dkk-1: Peptide 1 (a.a.71-85)—GNKYQTIDNYQPYPC (SEQ ID NO:118), Peptide 2 (a.a.165-186)—LDGYSRRTTLSSKMYHTKGQEG (SEQ ID NO:119), Peptide 3 (a.a.246-266)—RIQKDHHQASNSSRLHTCQRH (SEQ ID NO:120), Peptide 4 (a.a.147-161)—RGEIEETITESFGND (SEQ ID NO:121), and Peptide 5(232-250)—EIFQRCYCGEGLSCRIQKD (SEQ ID NO:122) of human Dkk-1. FIG. 26shows the location of the various peptides selected, their relationshipto the Dkk-1 amino acid sequence and polyclonal antibodies generated.

[0499] Western blots demonstrated that the antibodies generated againstpeptides 2 (Antibody #5521) (FIG. 27) and 4 (Antibody #74397) (FIG. 28)are specific toward Dkk-1. FIG. 27 shows Western blots using 500 μl ofconditioned medium (CM) from non-transfected 293 cells or from 293 cellstransfected with Dkk1 -V5 that were immunoprecipitated by anti-V5antibody. Bead elutes were separated by non-reducing SDS-PAGE (lanes #4,5 of FIG. 27). 20 μl of conditioned medium from both samples (lanes #2,3 of FIG. 27) and from Dkk1-AP transfected 293 cells (lane #6 of FIG.27) were additionally separated on the gel. The Western was performedusing antibodies Anti-V5/AP (1:10,000) and Ab#5521 (10 μg/ml). Ab#5521detected Dkk1-V5 and Dkk1-AP from conditioned medium.

[0500]FIG. 28 shows Western blot results using Ab#74397. Anti-V5/AP wastested at a 1:4000 dilution and Ab#74397 was tested at a 1:500 dilution.Ab#74397 was able to detect Dkk1-V5 in both conditioned medium andimmunoprecipitated conditioned medium.

[0501] The results obtained with antibodies #5521 and #74397 aresummarized in the following table: Rabbit Peptide Peptide PurifiedImmuno- No. Position Sequence (Y/N) Western precipitation Location 5521165-186 LDGYSR Y (Protein Y N/A Between RTTLSSK G Cy1 and MYHTKGpurified) Cys2 QEG domain 74397 147-161 RGEIEETI N Y N/A Between TESFGNCyl and D Cys2 domain

Example 6 Effects of Exogenous Dkk-1 on Wnt-Mediated Signaling in theXenopus Embryo Assay

[0502] Xenopus embryos are an informative and well-established in vivoassay system to evaluate the modulation of Wnt signaling (McMahon etal., Cell 58: 1075-84 (1989); Smith and Harland, 1991; reviewed inWodarz and Nusse 1998).

[0503] Modification of the Wnt signaling pathway can be visualized byexamining the embryos for a dorsalization phenotype (duplicated bodyaxis) after RNA injection into the ventral blastomere at the 4- or8-cell stage. On the molecular level, phenotypes can be analyzed bylooking for expression of various marker genes in stage 10.5 embryos.Such markers would include general endoderm, mesoderm, and ectodermmarkers as well as a variety of tissue-specific transcripts.

[0504] Analysis can be done by RT-PCR/TaqMan® and can be done on wholeembryo tissue or in a more restricted fashion (microdissection). Becausethis system is very flexible and rapid, by injecting combinations oftranscripts, such as HBM and different Wnts or Wnt antagonists, themechanism of HBM in the Wnt pathwaycan thereby be dissected.Furthermore, investigations are conducted to determine whether Zmax/LRP5and HBM differentially modulate Wnt signaling either alone, or incombination with other components. Previous studies have demonstratedthat LRP6 alone or LRP5+Wnt5a were able to induce axis duplication(dorsalization) in this system (Tamai et al., Nature 407: 530-35(2000)).

[0505] Constructs for Xenopus Expression (Vector pCS2⁺)

[0506] Constructs were prepared using the vector pCS2⁺. DNA inserts weresubcloned in the sense orientation with respect to the vector SP6promoter. The pCS2⁺ vector contains an SV40 virus polyadenylation signaland T3 promoter sequence (for generation of antisense mRNA) downstreamof the insert.

[0507] Full length Zmax/LRP5 and HBM ORF cDNA: Insert cDNA was isolatedfrom the full length cDNA retrovirus constructs (with optimized Kozaksequences) by BglII-EcoRI digestion and subcloned into the BamHI-EcoRIsites of the pCS2⁺ vector.

[0508] Full length XWnt8: This cDNA was PCR amplified from a Xenopusembryo cDNA library using oligos 114484 (SEQ ID NO:162)(5′-CAGTGAATTCACCATGCAAAACACCACTTTGTTC-3′) and 114487 (SEQ ID NO:163)(5′-CAGTTGCGGCCGCTCATCTCCGGTGGCCTCTG-3′). The oligos were designed toamplify the ORF with a consensus Kozak sequence at the 5′ end asdetermined from GenBank #X57234. PCR was carried out using the followingconditions: 96° C., 45 sec.; 63° C., 45 sec.; 72° C., 2 min. for 30cycles. The resulting PCR product was purified, subcloned intopCRII-TOPO (Invitrogen Corp.), sequence verified, and digested withBamHI/Xhol. This insert was subcloned into the vector at the BamHI-Xholsites.

[0509] Full length Wnt5a: A murine Wnt5a cDNA clone was purchased fromUpstate Biotechnology (Lake Placid, N.Y.) and subcloned into the EcoRIsite of the vector. Sequencing confirmed insert orientation.

[0510] Full length human Dkk-1: A human cDNA with GenBank accessionnumber AF127563 was available in the public database. Using thissequence, PCR primers were designed to amplify the open reading framewith a consensus Kozak sequence immediately upstream of the initiatingATG. Oligos 117162 (SEQ ID NO:164)(5′-CAATAGTCGACGAATTCACCATGGCTCTGGGCGCAGCGG-3′) and 117163 (SEQ IDNO:165) (5′-GTATTGCGGCCGCTCTAGATTAGTGTCTCTGACAAGTGTGAA-3′) were used toscreen a human uterus cDNA library by PCR. The resulting PCR product waspurified, subcloned into pCRII-TOPO (Invitrogen Corp.), sequenceverified, and digested with EcoRI/Xhol. This insert was subcloned intothe pCS2⁺ vector at the EcoRI-Xhol sites.

[0511] Full length human Dkk-2: A full length cDNA encoding human Dkk-2was isolated to investigate the specificity of the Zmax/LRP5/HBMinteraction with the Dkk family of molecules. Dkk-1 was identified inyeast as a potential binding partner of Zmax/LRP5/HBM. Dkk-1 has alsobeen shown in the literature to be an antagonist of the Wnt signalingpathway, while Dkk-2 is not (Krupnik et a., 1999). The Dkk-2 full lengthcDNA serves as a tool to discriminate the specificity and biologicalsignificance of Zmax/LRP5/HBM interactions with the Dkk family (e.g.,Dkk-1, Dkk-2, Dkk-3, Dkk-4, Soggy, their homologs and variant, etc.). Ahuman cDNA sequence for Dkk-2 (GenBank Accession No. NM_(—)014421) wasavailable in the public database. Using this sequence, PCR primers weredesigned to amplify the open reading frame with a consensus Kozaksequence immediately upstream of the initiating ATG. Oligos 51409 (SEQID NO:166) (5′-CTAACGGATCCACCATGGCCGCGTTGATGCGG-3′) and 51411 (SEQ IDNO:167) (5′-GATTCGAATTCTCAAATTTTCTGACACACATGG-3′) were used to screenhuman embryo and brain cDNA libraries by PCR. The resulting PCR productwas purified, subcloned into pCRII-TOPO, sequence verified, and digestedwith BamHI/EcoRI. This insert was subcloned into the pCS2⁺vector at theBamHI-EcoRI sites.

[0512] Full length LRP6 was isolated from the pED6dpc4 vector byXhoI-XbaI digestion. The full length cDNA was reassembled into theXhoI-XbaI sites of pCS2⁺. Insert orientation was confirmed by DNAsequencing.

[0513] mRNA Synthesis and Microinjection Protocol

[0514] mRNA for microinjection into Xenopus embryos is generated by invitro transcription using the cDNA constructs in the pCS2⁺ vectordescribed above as template. RNA is synthesized using the AmbionmMessage mMachine high yield capped RNA transcription kit (Cat. #1340)following the manufacturer's specifications for the Sp6 polymerasereactions. RNA products were brought up to a final volume of 50 μl insterile, glass-distilled water and purified over Quick Spin Columns forRadiolabelled RNA Purification G50-Sephadex (Roche, Cat. #1274015)following the manufacturer's specifications. The resulting eluate wasfinally extracted with phenol:chloroform:isoamyl alcohol and isopropanolprecipitated using standard protocols (Sambrook et al., 1989). Final RNAvolumes were approximately 50 μl. RNA concentration was determined byabsorbance values at 260 nm and 280 nm. RNA integrity was visualized byethidium bromide staining of denaturing (formaldehyde) agarose gelelectrophoresis (Sambrook et al., 1989). Various amounts of RNA (2 pg to1 ng) are injected into the ventral blastomere of the 4- or 8-cellXenopus embryo. These protocols are described in Moon et al.,Technique-J. of Methods in Cell and Mol. Biol. 1: 76-89 (1989), andPeng, Meth. Cell. Biol. 36: 657-62 (1991).

[0515] Screening for Duplicated Body Axis

[0516] In vitro transcribed RNA is purified and injected into a ventralblasomere of the 4- or 8-cell Xenopus embryo (approx. 2 hourspost-fertilization). At stage 10.5 (approx. 11 hourspost-fertilization), the injected embryos are cultured for a total of 72hours and then screened for the presence of a duplicated body axis(dorsalization) (FIG. 7). Using XWnt8-injected (2-10 pg) as a positivecontrol (Christian et al. (1991)) and water-injected or non-injectedembryos as negative controls, we replicated the published observationthat Zmax(LRP5)+Wnt5a (500 and 20 pg, respectively) could induce axisduplication. Wnt5a (20 pg) alone could not induce axis duplication (aspreviously reported by Moon et al. (1993)). We have also injected GFPRNA (100-770 pg) as a negative control to show that the amount of RNAinjected is not perturbing embryo development (not shown). Strikingly,HBM+Wnt5a (500 and 20 pg, respectively) yielded an approximately 3.5fold more robust response of the phenotype (p=0.043 by Fisher's exacttest) compared to Zmax(LRP5)+Wnt5a, suggesting that the HBM mutation isactivating the Wnt pathway (FIGS. 8 and 9). The HBM/Wnt5a embryos alsoappear to be more “anteriorized” than the Zmax(LRP5)/Wnt5a embryos,again suggestive of a gain-of-function mutation.

[0517] The role of Dkk-1 as a modulator of Zmax/LRP5- and HBM-mediatedWnt signaling was investigated. Literature reports have previouslycharacterized Xenopus and murine Dkk-1 as antagonists of the canonicalWnt pathway in the Xenopus system (Glinka et al., Nature 391:357-362(1998)). Using the human Dkk-1 construct, a dose-response assay wasperformed to confirm that our construct was functional and to identifythe optimal amount of RNA for microinjection. Using 250 pg/embryo ofhDkk-1 RNA, over 90% (p<0.001) of the embryos were observed to displayenlarged anterior structures (big heads) as anticipated from thepublished reports (FIG. 10).

[0518] The mechanism of hDkk-1 modulation of Wnt signaling in thepresence of Zmax/LRP5 or HBM was also investigated. Without any hDkk-1present, it was confirmed that HBM+Wnt5a was a more potent activator ofWnt signaling than Zmax/LRP5+Wnt5a (p<0.05). Interestingly, in thepresence of hDkk-1 (250 pg), Zmax/LRP5-mediated Wnt signaling wasrepressed (p<0.05) but hDkk-1 was unable to repress HBM-mediated Wntsignaling (p<0.01) (FIG. 11). The specificity of this observation can befurther addressed by investigating other members of the Dkk family,other Wnt genes, LRP6, additional Zmax/LRP5 mutants, and the peptideaptamers.

Example 7 Effects of Exogenous Dkk and LRP5 on Wnt Signaling in theTCF-Luciferase Assay

[0519] Wnt activity can be antagonized by many proteins includingsecreted Frizzled related proteins (SFRPs), Cerberus, Wnt InhibitoryFactor-1 and Dkk-1 (Krupnik et al., 1999). The Dkk family of proteinsconsists of Dkk-14 and Soggy, a Dkk-3-like protein. Dkk-1 and Dkk4 havebeen shown to antagonize Wnt mediated Xenopus embryo development,whereas Dkk-2, Dkk-3, and Soggy do not. Unlike many of these proteinsthat antagonize Wnt activity by directly interacting with Wnt proteins,Dkk-1 acts by binding to two recently identified Wnt coreceptors, LRP5and LRP6. (Mao et al., 2001; Bafico et al., 2001). The details of thisinteraction have been examined by the present inventors and Mao et al.using deletion constructs of LRP6, which demonstrated that EGF repeats 3and 4 are important for Dkk-1 interaction. Accordingly, the activity oftwo Dkk proteins, Dkk-1 and Dkk-2, were investigated with various Wntmembers, LRP5, LRP6, and the mutant form of LRP5, designated HBM. Thepresent invention explores whether there is any functional differencebetween LRP5 and HBM with regard to Dkk action on Wnt mediatedsignaling. Various reagents were developed, including Dkk-1 peptides,constrained LRP5 peptide aptamers, constrained Dkk-1 peptide aptamersand polyclonal antibodies to Dkk-1 (in Example 5 above) to identifyfactors that mimic HBM mediated Wnt signaling.

[0520] Methods

[0521] Various LRP5 constrained peptides were developed. Specifically,four peptides that interact with the LBD of LRP5 (FIG. 4 constructsOST259-262 in FIG. 12) and three peptides that interact with thecytoplasmic domain of LRP5 (constructs OST266-OST268 in FIG. 12). Inaddition two Dkk-1 peptides were developed: constructs OST264 and OST265in FIG. 12, corresponding to Dkk-1 amino acids 139-266 and 96-245,containing the smallest region of Dkk-1 that interacts with LRP5 (FIG.6). The cDNA clones encoding the LRP5 LBD interacting peptides and theDkk-1 peptides were subcloned into pcDNA3.1 with the addition of a Kozakand signal sequence to target the peptide for secretion. The constructsencoding the three peptides interacting with the cytoplasmic domain ofLRP5 were also subcloned into pcDNA3.1. However, these latter constructsdo not contain a signal sequence.

[0522] HOB-03-CE6 osteoblastic cells developed by Wyeth Ayerst(Philadelphia, Pa.) were seeded into 24-well plates at 150,000 cells perwell in 1 ml of the growth media (D-MEM/F12 phenol red-free) containing10% (v/v) heat-inactivated FBS, 1× penicillin streptomycin, and 1×Glutamax-1, and incubated overnight at 34° C. The following day, thecells were transfected using Lipofectamine 2000® (as described by themanufacturer, Invitrogen) in OptiMEM (Invitrogen) with 0.35 μg /well ofLRP5, HBM, or control plasmid DNA (empty vector pcDNA3.1) and eitherWnt1 or Wnt3a plasmid DNA. Similar experiments were performed with LRP6plasmid DNA (0.35 μg/well) or a control pEDdpc4 empty vector.Furthermore, each of these groups were then divided into three groups,those receiving 0.35 μg/well Dkk-1, Dkk-2, or pcDNA3.1 control DNA. Allwells were transfected with 0.025 μg/well of CMV beta-galactosidaseplasmid DNA and 0.35 μg/well 16× TCF(AS)-luciferase reporter DNA(developed by Ramesh Bhat, Wyeth-Ayerst (Philadelphia, Pa.)). After 4hours of incubation, the cells were rinsed and 1 ml of fresh growthmedia was added to each well. The cells were cultured overnight at 34°C., followed by a wash and a change of media. Cells were cultured for anadditional 18-24 hours at 37° C. Cells were then lysed with 50 μl/wellof 1× lysis buffer. The extracts were assayed for beta-galactosidaseactivity (Galacto Reaction Buffer Diluent & Light Emission Accelerator,Tropix) using 5 μl extract+50 μl beta-galactosidase diluent andluciferase activity (Luciferase Assay Reagent, Promega) using 20 μlextract.

[0523] U2OS human osteosarcoma cells were also utilized. U2OS cells(ATCC) were seeded into 96-well plates at 30,000 cells per well in 200μl of the growth media (McCoy's 5A) containing 10% (v/v)heat-inactivated FBS, 1× penicillin streptomycin, and 1× Glutamax-1, andincubated overnight at 37° C. The following day, the media was replacedwith OptiMEM (Invitroge) and cells were transfected using Lipofectamine2000® (as described by the manufacturer, Invitrogen) with 0.005 μg/wellof LRP5, HBM, LRP6 or contol plasmid DNA (empty vector pcDNA3.1) andeither Wnt1 (0.0025 μg/well) or Wnt3a (0.0025 ug/well) plasmid DNA. Inaddition, the 16x-(AS) TCF-TK-firefly-luciferase (Ramesh Bhat, WHRI,Wyeth) and control TK-renilla luciferase (Promega Corp.) wereco-transfected at 0.3 ug/well and 0.06 ug/well respectively in allexperiments. Futhermore, each of these groups was then divided intodifferent groups, those receiving 0.05 ug/well Dkk-1, Dkk-2, Dkk3, Dkk1-Alkaline Phosphatase (AP), mutant Dkk-1 (C220A), Soggy or pcDNA3.1control DNA. In other experiments, cells were co-transfected with 0.005pg/well of LRP5, 0.0025 ug/well of Wnt1 or Wnt3a (using 0.0025 pg/wellof a control pcDNA3.1) with LRP5-interacting aptamers (0.05 ug/well).Cells were cultured for an additional 18-20 hours at 37° C. Culturemedium was removed. Cells were cultured for an additional 18-20 hours at37° C. Culture medium was removed. Cells were then lysed with 100μl/well of 1× Passive Lysis Buffer (PLB) of Dual Luciferase Reagent kit(DLR-kit-Promega Corp.) 20 μl of the lysates were combined with LARIIreagent of DLR-kit and assayed for TCF-firefly luciferase signal in TopCount (Packard) instrument. After measuring the Firefly readings, 100 μlof the “Stop and Glo” reagent of DLR kit that contains a quencher and asubstrate for renilla luciferase was added into each well. Immediatelythe renilla luciferase reading was measured using the Top Count(Packard) Instrument. The ratios of the TCF-firefly luciferase tocontrol renilla readings were calculated for each well and the meanratio of triplicate or more wells was expressed in all data.

[0524] Results

[0525] The results of these experiments demonstrate that Dkk-1, in thepresence of Wnt1 and LRP5, significantly antagonized TCF-luciferaseactivity (FIG. 14). In marked contrast, Dkk-1 had no effect on HBM/Wnt1mediated TCF-luciferase activity (FIG. 14). In similar experiments,Dkk-1 was also able to antagonize LRP5/Wnt3a but not HBM/Wnt3a mediatedTCF-luciferase activity (FIG. 15). These results indicate that the HBMmutation renders Dkk-1 inactive as an antagonist of Wnt1 and Wnt3asignaling in HOB03CE6 osteoblastic cells. In other experiments withWnt1, Dkk-1 had no effect on LRP5 or HBM mediated TCF-luciferaseactivity (FIG. 14). In contrast, with either LRP5 or HBM in the presenceof Wnt3a, Dkk-2 was able to antagonize the TCF-luciferase activity (FIG.15). These latter results indicate that the HBM mutation has no effecton Dkk-2 action in the presence of Wnt3a. Experiments were alsoperformed using the closely related LRP6 cDNA in HOB-03-CE6 cells. Inthese experiments, LRP6/Wnt1 and LRP6/Wnt3a mediated TCF-luciferase wereregulated in the same manner as LRP5. Specifically, Dkk-1 antagonizedLRP6/Wnt1 mediated TCF-luciferase activity, whereas Dkk-2 had no effect(FIG. 14). However, similar to the action of Dkk-2 with LRP5/Wnt3a,Dkk-2 was able to antagonize LRP6/Wnt3a mediated TCF-luciferase activity(FIG. 15).

[0526] The results in the U2OS cells show a robust effect of the OST262LRP5 peptide aptamer activation of Wnt signaling in the presence ofWnt3a (FIG. 16). These functional results are confirmed by the resultsshown below in Example 11 using LRP5 peptide aptamers in the Xenopusassay. Such results affirmatively demonstrate that the effects of smallmolecules on LRP5/LRP6/HBM signaling can be detected using theTCF-luciferase assay.

[0527] These data demonstrate that there is a functional differencebetween LRP5 and HBM regarding the ability of Dkk-1 to antagonize Wnt1and Wnt3a signaling. These data and previous data showing that Dkk-1directly interacts with LRP5 suggests that the inability of Dkk-1 toantagonize HBM/Wnt signaling may in part contribute to the HBMphenotype. These experiments further demonstrate the ability to testvarious molecules (e.g., small molecules, aptamers, peptides,antibodies, LRP5 interacting proteins or Dkk-1 interacting proteins, andthe like) for a LRP5 ligand that mimics HBM mediated Wnt signaling orfactors that block Dkk-1 interaction with LRP5.

Example 8 Yeast-2 Hybrid Interaction Trap

[0528] Small molecule inhibitors (or partial inhibitors) of the Dkk-LRPinteraction may be an excellent osteogenic therapeutic. One way toinvestigate this important protein-protein interaction is using Y2Htechniques substantially as described above and as is well known in theart. Regions of LRP5, such as LRP5 LBD, have been found to functionallyinteract with Dkk. This interaction is quantitated using a reporterelement known in the art, e.g., LacZ or luciferase, which is onlyactivated when bait and prey interact. The Y2H assay is used to screenfor compounds which modulate the LRP-Dkk interaction. Such a modulationwould be visualized by a reduction in reporter element activationsignifying a weaker or disrupted interaction, or by an enhancement ofthe reporter element activation signifying a stronger interaction. Thus,the Y2H assay can be used as a high-throughput screening technique toidentify compounds which disrupt or enhance Dkk interaction withLRP5/LRP6/HBM, which may serve as potential therapeutics.

[0529] For example, the Interaction Trap methodology can be used asfollows. The LRP5 LBD, for example, was fused with LexA and Dkk-1 wasfused with either Gal4-AD or B42. With the LRP5LBD-LexA bait and theGal4AD-Dkk prey, over a 20-fold activation of a lacZ reporter (under thecontrol of a single LexA operator) was detected over the background.Using a Dkk-1 mutant (C220A) that is unable to bind to LRP, theinteraction was reduced in yeast, showing the specificity of thisinteraction and system (FIG. 18). As a result, small molecules may beidentified that modulate this interaction between LRP and Dkk.

Example 9 Cell-Based Functional High-Throughput Assay

[0530] To develop a high throughput assay, the TCF-luciferase assaydescribed in Example 7 was modified utilizing low level expression ofendogenous LRP5/6 in U2OS and HEK293 cells. However, HOB-03-CE6 cellsand any other cells which show a differential response to Dkk dependingon whether LRP5, LRP6 or HBM are expressed. Using U2OS (humanosteosarcoma) and HEK293 (ATCC) cells, the TCF-luciferase and tk-Renillareporter element constructs were co-transfected along with Wnt3a/l andDkk. Wnt3a alone, by using endogenous LRP5/6, was able to stimulate TCFreporter gene activation. When Dkk, is co-transfected with Wnt3a/Wnt 1and reporters (TCF-luci and tk-Renilla), Dkk represses reporter elementactivity. In addition, the TCF-luci signal is activated by Wnt3a/Wnt1can be repressed by the addition of Dkk-enriched conditioned media tothe cells containing Wnt3a/Wnt1 and reporters. The assay is furthervalidated by the lack of TCF-reporter inhibition by a point mutantconstruct (C220A) of Dkk1.

[0531] The Dkk-mediated repression of the reporter is dependent upon theconcentration of transfected Dkk cDNA or on the amount ofDkk-conditioned media added. In addition, the Dkk-mediated reportersuppression can be altered by the co-transfection of LRP5, LRP6, and HBMcDNAs in the U2OS or HEK293 cells. In general, U2OS cells show greatersensitivity to Dkk-mediated reporter suppression than that in HEK-293cells. In U2OS cells, the transfection of LRP5/LRP6/HBM cDNA leads tomoderate activation of TCF-luci in the absence of Wnt3a/Wnt1transfection. This activation presumably utilizes the endogenous Wntspresent in U2OS cells. Under this condition, Dkk1 can repress TCF-luciand shows a differential signal between LRP5 and HBM. By co-transfectingWnt3a/Wnt1, there is a generalized increase in the TCF-luci signal inthe assay. Further, one can detect Dkk-mediated differential repressionof the reporter due to LRP5 and HBM cDNA expression as well as betweenLRP5 and LRP6 cDNA. The repression is maximal with LRP6, moderate withLRP5, and least with HBM cDNA expression. In addition, the assay candetect the functional impact of the LRP5 interacting peptide aptamers(FIG. 4), Dkk1 interacting aptamers and binding domains of Dkk-1 (FIG.6; OST264 and OST265 of FIGS. 12 and 13).

[0532] Using this system with a suppressed Wnt-TCF signal due to thepresence of both Dkk and Wnt3a, one can screen for compounds that couldalter Dkk modulation of Wnt signaling, by looking for compounds thatactivate or the TCF-luciferase reporter, and thereby relieve theDkk-mediated repression of the Wnt pathway. Such compounds identifiedmay potentially serve as HBM-mimetics and be useful, for example, asosteogenic therapeutics. Data generated from this high throughput screenare demonstrated in FIGS. 19-21. FIG. 19 shows that Dkk1 repressesWnt3a-mediated signaling in U2OS bone cells. FIG. 20 demonstrates thefunctional differences between LRP5, LRP6, and HBM. Dkk-1 represses LRP6and LRP5 but has little or no effect on HBM-generated Wnt1 signaling inU2OS cells. FIG. 21 demonstrates the differential effects of various Dkkfamily members and modified Dkks, including Dkk-1, a mutated Dkk-1(C220A), Dkk-1-AP (modified with alkaline phosphatase), Dkk-3, andSoggy.

Example 10 DKK/LRP5/6/HBM ELISA Assay

[0533] A further method to investigate Dkk binding to LRP is via ELISAassay. Two possible permutations of this assay are exemplified. LRP5 isimmobilized to a solid surface, such as a tissue culture plate well. Oneskilled in the art will recognize that other supports such as a nylon ornitrocellulose membrane, a silicon chip, a glass slide, beads, etc. canbe utilized. In this example, the form of LRP5 used is actually a fusionprotein where the extracellular domain of LRP5 is fused to the Fcportion of human IgG. The LRP5-Fc fusion protein is produced in CHO cellextracts from stable cell lines. The LRP5-Fc fusion protein isimmobilized on the solid surface via anti-human Fc antibody or byProtein-A or Protein G-coated plates, for example. The plate is thenwashed to remove any non-bound protein. Conditioned media containingsecreted Dkk protein or secreted Dkk-epitope tagged protein (or purifiedDkk or purified Dkk-epitope tagged protein) is incubated in the wellsand binding of Dkk to LRP is investigated using antibodies to either Dkkor to an epitope tag. Dkk-V5 epitope tagged protein would be detectedusing an alkaline phosphatase tagged anti-V5 antibody.

[0534] Alternatively, the Dkk protein could be directly fused to adetection marker, such as alkaline phosphatase. Here the detection ofthe Dkk-LRP interaction can be directly investigated without subsequentantibody-based experiments. The bound Dkk is detected in an alkalinephosphatase assay. If the Dkk-alkaline phosphatase fusion protein isbound to the immobilized LRP5, alkaline phosphatase activity would bedetected in a colorimetric readout. As a result, one can assay theability of small molecule compounds to alter the binding of Dkk to LRPusing this system. Compounds, when added with Dkk (or epitope-taggedDkk) to each well of the plate, can be scored for their ability tomodulate the interaction between Dkk and LRP based on the signalintensity of bound Dkk present in the well after a suitable incubationtime and washing. The assay can be calibrated by doing cold competitionexperiments with unlabeled Dkk or with a second type of epitope-taggedDkk. Any small molecule that is able to modulate the Dkk-LRP interactionmay be a suitable therapeutic candidate, more preferably an osteogenictherapeutic candidate.

Example 11 Functional Evaluation of Peptide Aptamers in Xenopus

[0535] The constrained peptide aptamers constructs OST258-263 (where 258contains the signal sequence by itself and 263 contains an irrelevantconstrained peptide) (FIGS. 12 and 13) were used to generate RNAsubstantially as described in Example 7, except the vector waslinearized by restriction endonuclease digestion and RNA was generatedusing T7 RNA polymerase.

[0536] Aptamer RNA was injected at 250 pg per blastomere using theprotocol of Example 7. Wnt signaling was activated, as visualized byembryo dorsalization (duplicated body axis) with aptamers 261 and, morestrongly, 262. The results of this assay are shown in FIGS. 22 and 23.These results suggest that aptamers 261 and 262 are able to activate Wntsignaling possibly by binding to the LBD of LRP, thereby preventing themodulation of LRP-mediated signaling by Dkk.

[0537] The aptamers of the present invention can serve as HBM-mimetics.In the Xenopus system they are able to induce Wnt signaling all bythemselves. They may also serve as tools for rational drug design byenhancing the understanding of how peptides are able to interact withLRP and modulate Wnt signaling at the specific amino acid level. Thus,one would be able to design small molecules to mimic their effects astherapeutics. In addition, the aptamers identified as positives in thisassay may be used as therapeutic molecules themselves.

Example 12 Homogenous Assay

[0538] An excellent method to investigate perturbations inprotein-protein interactions is via Fluorescence Resonance EnergyTransfer (FRET). FRET is a quantum mechanical process where afluorescent molecule, the donor, transfers energy to an acceptorchromophore molecule which is in close proximity. This system has beensuccessfully used in the literature to characterize the intermolecularinteractions between LRP5 and Axin (Mao et al., Molec. Cell Biol.7:801-809). There are many different fluorescent tags available for suchstudies and there are several ways to fluorescently tag the proteins ofinterest. For example, CFP (cyan fluorescent protein) and YFP (yellowfluorescent protein) can be used as donor and acceptor, respectively.Fusion proteins, with a donor and an acceptor, can be engineered,expressed, and purified.

[0539] For instance, purified LRP protein, or portions or domainsthereof, fused to CFP and purified Dkk protein, or portions or domainsthereof that interact with Dkk or LRP respectively, fused to YFP can begenerated and purified using standard approaches.

[0540] If LRP-CFP and Dkk-YFP are in close proximity, the transfer ofenergy from CFP to YFP will result in a reduction of CFP emission and anincrease in YFP emission.

[0541] Energy is supplied with an excitation wavelength of 450 nm andthe energy transfer is recorded at emission wavelengths of 480 nm and570 nm. The ratio of YFP emission to CFP emission provides a guage forchanges in the interaction between LRP and Dkk. This system is amenablefor screening small molecule compounds that may alter the Dkk-LRPprotein-protein interaction. Compounds that disrupt the interactionwould be identified by a decrease in the ratio of YFP emission to CFPemission. Such compounds that modulate the LRP-Dkk interaction wouldthen be considered candidate HBM mimetic molecules. Furthercharacterization of the compounds can be done using the TCF-luciferaseor Xenopus embryo assays to elucidate the effects of the compounds onWnt signaling.

[0542] While the above example describes a cell-fee, solution-phaseassay using purified components, a similar cell-based assay could alsobe performed. For example, LRP-CFP fusion protein can be expressed incells. The Dkk-YFP fusion protein then could be added to the cellseither as purified protein or as conditioned media. The interaction ofLRP and Dkk is then monitored as described above.

[0543] All references cited herein are hereby incorporated by referencein their entirety for all purposes. The following applications are alsoincorporated by reference in their entirety herein for all purposes:U.S. Application No. 60/290,071, filed May 11, 2001;

[0544] U.S. application Ser. No. 09/544,398, filed on Apr. 5, 2000; U.S.application Ser. No. 09/543,771, filed Apr. 5, 2000; 09/578,900; U.S.application Ser. No. 09/229,319, filed Jan. 13, 1999; U.S. ProvisionalApplication 60/071,449, filed Jan. 13, 1998; and

[0545] International Application PCT/US00/16951, filed Jun. 21, 2000;International PCT Application entitled “HBM Variants That Modulate BoneMass and Lipid Levels,” filed May 13, 2002; and International PCTApplication entitled “Transgenic Animal Model of Bone Mass Modulation,”filed May 13, 2002. Additionally, this application claims priority toU.S. provisional applications 60/291,311, filed May 17, 2001;60/353,058, filed Feb. 1, 2002; and 60/361,293, filed Mar. 4, 2002; thetexts of which are herein incorporated by reference in their entiretyfor all purposes.

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040038860). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

We claim:
 1. A method of regulating LRP5, LRP6, or HBM activity in asubject comprising administering a composition which modulates a Dkkactivity in an amount effective to regulate LRP5, LRP6, or HBM activity.2. The method of any of claims 1, 24, 28, 33, 36, 37, 48, 64, 65, 93,98, 101, 105, 107, 111, or 112, wherein the Dkk is Dkk-1.
 3. The methodof any of claims 1, 24, 28, or 33, wherein the Dkk is Dkk-1 and the Dkkactivity is inhibited.
 4. The method of claims 1 or 24, wherein the Dkkactivity modulates bone mass and/or lipid levels.
 5. The method of claim4, wherein bone mass is increased and/or lipid levels are decreased. 6.The method of claim 5, wherein the increase in bone mass is determinedvia one or more of a decrease in fracture rate, an increase in bonestrength, an increase in bone density, an increase in bone mineraldensity, an increase in trabecular connectivity, an increase intrabecular density, an increase in cortical density, an increase in bonediameter, and an increase in inorganic bone content.
 7. The method ofany of claims 1, 24, 28, or 33, wherein said composition comprises oneor more compounds selected from the group consisting of Dkk interactingproteins, or a Dkk-binding fragment thereof.
 8. The method of any ofclaims 1, 24, 28, or 33, wherein said composition comprises anantisense, a siRNA, or shRNA molecule which recognizes and binds to anucleic acid encoding one or more Dkk interacting proteins.
 9. Themethod of any of claims 1, 24, 28, or 33, and wherein said compositioncomprises a Dkk peptide aptamer.
 10. The method of any of claims 1, 24,28, or 33, wherein said composition comprises a mimetic of a Dkk peptideaptamer.
 11. The method of any of claims 1, 24, 28, or 33, wherein saidcomposition inhibits Dkk binding to LRP5, LRP6, or HBM.
 12. The methodof any of claims 1, 24, 28, or 33, wherein said composition enhancesbinding of Dkk to LRP5, LRP6, or HBM.
 13. The method of any of claims 1,24, 28, or 33, wherein said composition comprises a Dkk interactingprotein peptide aptamer.
 14. The method of any of claims 1, 24, 28, or33, wherein said composition comprises a mimetic of a Dkk interactingprotein peptide aptamer.
 15. The method of any of claims 1, 24, 28 or33, wherein said composition inhibits Dkk interacting protein orDkk-binding fragment thereof binding to Dkk.
 16. The method of any ofclaims 1, 24, 28, or 33, wherein said composition enhances binding ofDkk interacting protein or Dkk-binding fragment thereof to Dkk.
 17. Themethod of any of claims 1, 24, 28, or 33, wherein said subject is avertebrate or an invertebrate organism.
 18. The method of any of claims1, 24, 28, or 33, wherein said subject is a mammal.
 19. The method ofany of claims 1, 24, 28, or 33, wherein said subject is a canine, afeline, an ovine, a primate, an equine, a porcine, a caprine, a camelid,an avian, a bovine, or a rodent.
 20. The method of claim 19, whereinsaid primate is a human.
 21. The method of any of claims 1, 24, 28, or33, wherein said composition comprises an LRP5 peptide aptamer.
 22. Themethod of claim 21, wherein said peptide aptamer is OST262 (SEQ IDNO:208).
 23. The method of any of claims 1, 24, 28 or 33, wherein thecomposition comprises an LRP5 antibody or an immunologically activefragment thereof.
 24. A method of regulating Dkk-Wnt pathway activity ina subject comprising administering a composition which modulates Dkkactivity in an amount effective to regulate Dkk-Wnt pathway activity.25. The method of claims 24, 101, or 107, wherein the Wnt is one or moreof Wnt1-Wnt19.
 26. The method of claim 25, wherein the Wnt is Wnt1,Wnt3, Wnt3a, or Wnt10b.
 27. The method of claim 24 wherein saidcomposition which modulates Dkk activity or modulates Dkk interactionwith LRP5/LRP6/HBM is administered in an amount effective to modulateWnt signaling.
 28. A method of modulating bone mass in a subjectcomprising administering to the subject a composition which modulatesDkk activity or Dkk interaction with LRP5, LRP6, or HBM in an amounteffective to modulate bone mass in the subject.
 29. The method of claim28, wherein bone mass is increased.
 30. The method of the previousclaim, wherein the increase in bone mass is determined via one or moreof a decrease in fracture rate, an increase in bone strength, anincrease in bone density, an increase in bone mineral density, anincrease in trabecular connectivity, an increase in trabecular density,an increase in cortical density, an increase in bone diameter, and anincrease in inorganic bone content.
 31. The method of claims 28 or 36,wherein said subject has a bone mass disorder selected from the groupconsisting of a bone development disorder, a bone fracture, age-relatedloss of bone, chrondrodystrophy, drug-induced bone disorder, high boneturnover, hypercalcemia, hyperostosis, osteogenesis imperfecta,osteomalacia, osteomyelitis, osteoporosis, Paget's disease,osteoarthritis, and rickets.
 32. The method of claim 28, wherein thecomposition which modulates Dkk activity or Dkk interaction with LRP5,LRP6, or HBM is administered in an amount effective to modulate theamount of trabecular and/or cortical tissue.
 33. A method of modulatinglipid levels in a subject comprising administering to the subject acomposition which modulates Dkk activity or Dkk interaction with LRP5,LRP6, or HBM in an amount effective to modulate lipid levels in thesubject.
 34. The method of claim 33, wherein lipid levels are decreased.35. The method of claim 33 or 36, wherein the subject has alipid-modulated disorder and wherein the lipid-modulated disorder isselected from the group consisting of a cardiac condition,atherosclerosis, familial lipoprotein lipase deficiency, familialapoprotein CII deficiency, familial type 3 hyperlipoproteinemia,familial hypercholesterolemia, familial hypertriglyceridemia, multiplelipoprotein-type hyperlipidemia, elevated lipid levels due to dialysisand/or diabetes, and elevated lipid levels of unknown etiology.
 36. Amethod of diagnosing low or high bone mass and/or high or low lipidlevels in a subject comprising examining expression of Dkk, LRP5, LRP6,HBM, or and HBM-like variant in the subject and determining whether Dkk,LRP5, LRP6, HBM, or an HBM-like variant is over- or under-expressed todetermine whether subject has (a) high or low bone mass and/or (b) hashigh or low lipid levels.
 37. A method of screening for a compound whichmodulates the interaction of Dkk with LRP5, LRP6, HBM, or a Dkk-bindingfragment of LRP5, LRP6, or HBM comprising: (a) exposing Dkk and a LRP5,LRP6, and/or HBM binding fragment thereof to a compound; and (b)determining whether said compound modulates Dkk interaction with theLRP5/LRP6/HBM binding fragment.
 38. The method of claim 37, wherein saidmodulation is determined by whether said compound binds to Dkk or theLRP5, LRP6, or HBM binding fragment thereof.
 39. The method of claim 37,wherein Dkk or a LRP-binding fragment thereof is attached to asubstrate.
 40. The method of claim 37, wherein said compound comprisesone or more compounds selected from the group consisting of Dkkinteracting proteins, or a Dkk-binding fragment thereof.
 41. The methodof claim 37 or 48, wherein said compound comprises a Dkk peptideaptamer.
 42. The method of claim 37 or 48, wherein said compoundcomprises a mimetic of a Dkk peptide aptamer.
 43. The method of claim 37or 48, wherein said compound comprises a Dkk interacting protein peptideaptamer.
 44. The method of claim 37 or 48, wherein the compoundcomprises an LRP5 peptide aptamer.
 45. The method of claim 44, whereinthe peptide aptamer is OST262 (SEQ ID NO:208).
 46. The method of claim37 or 48, wherein the compound comprises an LRP5 antibody.
 47. Themethod of claim 37 or 48, wherein said compound is a mimetic of a Dkkinteracting protein peptide aptamer.
 48. A method of screening for acompound which modulates the interaction of Dkk with a Dkk interactingprotein comprising: (a) exposing a Dkk interacting protein or aDkk-binding fragment thereof to a compound; and (b) determining whethersaid compound bound to a Dkk interacting protein or the Dkk-bindingfragment thereof; and (c) further determining whether said compoundmodulates the interaction of Dkk interacting protein and Dkk.
 49. Themethod of claim 48, wherein the Dkk interacting protein or a Dkk-bindingfragment thereof is attached to a substrate.
 50. A compositioncomprising a LRP5, LRP6, or HBM activity-modulating compound and apharmaceutically acceptable carrier therefor.
 51. The composition ofclaim 50, wherein said LRP5, LRP6, or HBM activity-modulating compoundcomprises a compound which binds to Dkk thereby modulating theinteraction of Dkk with LRP5, LRP6, or HBM.
 52. The composition of claim50, wherein said LRP5, LRP6, or HBM modulating compound comprises one ormore Dkk interacting proteins and Dkk-binding fragments thereof.
 53. Thecomposition of claim 50, wherein said LRP5, or LRP6, or HBM modulatingcompound is a monoclonal antibody or an immunologically active fragmentthereof which binds to a Dkk interacting protein, or a Dkk-bindingfragment thereof.
 54. The composition of claim 53, wherein themonoclonal antibody is human, chimeric, humanized, primatized®, orbispecific.
 55. The composition of claim 50, wherein said LRP5, LRP6, orHBM modulating compound comprises an antisense, a siRNA, or shRNAmolecule which recognizes and binds to a nucleic acid encoding one ormore Dkk interacting proteins.
 56. The composition of claim 50, whereinsaid LRP5, LRP6, or HBM modulating compound comprises a Dkk peptideaptamer.
 57. The composition of claim 50, wherein said LRP5, LRP6, orHBM modulating compound comprises a mimetic of a Dkk peptide aptamer.58. The composition of claim 50, wherein said LRP5, LRP6, or HBMmodulating compound comprises a Dkk interacting protein peptide aptamer.59. The composition of claim 50, wherein said LRP5, LRP6, or HBMmodulating compound comprises a mimetic of a Dkk interacting proteinpeptide aptamer.
 60. The composition of claim 50, wherein the compoundcomprises an LRP5 peptide aptamer.
 61. The composition of claim 60,wherein the peptide aptamer is OST262.
 62. The composition of claim 50,wherein the compound comprises an LRP5 antibody.
 63. A pharmaceuticalcomposition comprising a compound which modulates Dkk activity and apharmaceutically acceptable carrier therefor.
 64. A method foridentifying compounds which modulate Dkk and LRP5/LRP6/HBM interactionscomprising: (a) creating an LRP5, LRP6, or HBM fluorescent fusionprotein using a first fluorescent tag; and (b) creating a Dkk fusionprotein comprising a second fluorescent tag; (c) adding a test compound;and (d) assessing changes in the ratio of fluorescent tag emissionsusing Fluorescence Resonance Energy Transfer (FRET) or BioluminescenceResonance Energy Transfer (BRET) to determine whether the compoundmodulates Dkk and LRP5/LRP6/HBM interactions.
 65. A method ofidentifying binding partners for a Dkk protein comprising the steps of:(a) exposing the Dkk protein(s) or a LRP5/LRP6 binding fragment thereofto a potential binding partner; and (b) determining if the potentialbinding partner binds to a Dkk protein or the LRP5/LRP6 binding fragmentthereof.
 66. A nucleic acid encoding a Dkk interacting protein peptideaptamer comprising a nucleic acid encoding a scaffold protein in-framewith the activation domain of Gal4 or LexA that is in-frame with anucleic acid that encodes a Dkk interacting protein amino acid sequence.67. A vector comprising the nucleic acid of claim
 66. 68. The nucleicacid of claim 66, wherein the scaffold protein is trxA.
 69. A method ofdetecting a modulatory activity of a compound on the binding interactionof a first peptide and a second peptide of a peptide binding pair thatbind through extracellular interaction in their natural environment,comprising: (i) culturing at least one eukaryotic cell comprising: a) anucleotide sequence encoding a first heterologous fusion proteincomprising the first peptide or a segment thereof joined to atranscriptional activation protein DNA binding domain; b) a nucleotidesequence encoding a second heterologous fusion protein comprising thesecond peptide or a segment thereof joined to a transcriptionalactivation protein transcriptional activation domain; wherein binding ofthe first peptide or segment thereof and the second peptide or segmentthereof reconstitutes a transcriptional activation protein; and c) areporter element activated under positive transcriptional control of thereconstituted transcriptional activation protein, wherein expression ofthe reporter element produces a selected phenotype; (ii) incubating theeukaryotic cell in the presence of a compound under conditions suitableto detect the selected phenotype; and (iii) detecting the ability of thecompound to affect the binding interaction of the peptide binding pairby determining whether the compound affects the expression of thereporter element which produces the selected phenotype; wherein (1) saidfirst peptide is a Dkk peptide and the second peptide is a peptideselected from LRP5, HBM, LRP6 and the Dkk-binding portion ofLRP5/LRP6/HBM or (2) said first peptide is a Dkk interacting protein orthe Dkk-binding fragment thereof and said second peptide is a Dkkpeptide.
 70. The method of claim 69, wherein the eukaryotic cell is ayeast cell.
 71. The method of claim 70, wherein the yeast cell isSaccharomyces.
 72. The method of claim 71, wherein the Saccharomycescell is Saccharomyces cerevisiae.
 73. The method of claim 69, whereinthe Dkk is Dkk-1 and wherein the compound comprises one or more Dkkinteracting proteins or a Dkk-binding fragment thereof.
 74. The methodof claim 73, wherein the compound is directly added to assay.
 75. Themethod of claim 73, wherein the compound is recombinantly expressed bysaid eukaryotic cell in addition to said first and second peptides. 76.The method of claim 69, wherein the compound comprises a Dkk peptideaptamer.
 77. The method of claim 69, wherein the compound comprises amimetic of a Dkk peptide aptamer.
 78. The method of claim 69, whereinthe compound comprises a Dkk interacting protein peptide aptamer. 79.The method of claim 69, wherein the compound comprises a mimetic of aDkk interacting protein peptide aptamer.
 80. The method of claim 69,wherein the eukaryotic cell further comprises at least one endogenousnucleotide sequence selected from the group consisting of a nucleotidesequence encoding the DNA binding domain of a transcriptional activationprotein, a nucleotide sequence encoding the transcriptional activationdomain of a transcriptional activation protein, and a nucleotidesequence encoding the reporter element, wherein at least one of theendogenous nucleotide sequences is inactivated by mutation or deletion.81. The method of claim 69, wherein the peptide binding pair comprises aligand and a receptor to which the ligand binds.
 82. The method of claim69, wherein the transcriptional activation protein is Gal4, Gcn4, Hap1,Adr1, Swi5, Ste12, Mcm1, Yap1, Ace1, Ppr1, Arg81, Lac9, QalF, VP16, or amammalian nuclear receptor.
 83. The method of claim 69, wherein at leastone of the heterologous fusion proteins is expressed from anautonomously-replicating plasmid.
 84. The method of claim 69, whereinthe DNA binding domain is a heterologous DNA-binding domain of atranscriptional activation protein.
 85. The method of claim 84, whereinthe DNA binding protein is selected from the group consisting of amammalian steroid receptor and bacterial LexA protein.
 86. The method ofclaim 69, wherein the reporter element is selected from the groupconsisting of lacZ, a polynucleotide encoding luciferase, apolynucleotide encoding green fluorescent protein (GFP), and apolynucleotide encoding chloramphenicol acetyltransferase.
 87. Themethod of claim 86, wherein the reporter element is LacZ.
 88. The methodof claim 69, wherein the test sample comprises an LRP5 peptide aptamer.89. The method of claim 88, wherein the peptide aptamer is OST262 (SEQID NO:208).
 90. The method of claim 69, wherein the test samplecomprises an LRP5 antibody.
 91. A transgenic animal wherein Dkk-1 isknocked out in a tissue-specific fashion.
 92. The transgenic animal ofclaim 91, wherein the tissue specificity is bone tissue, cancer tissue,or liver tissue.
 93. A method for identifying potential compounds whichmodulate Dkk activity comprising: a) measuring the effect on binding ofone or more Dkk interacting proteins, or a Dkk-binding fragment thereof,with Dkk or a fragment thereof in the presence and absence of acompound; and b) identifying as a potential Dkk modulatory compound acompound which modulates the binding between one or more Dkk interactingproteins or Dkk-binding fragment thereof and Dkk or fragment thereof.94. A peptide aptamer of FIG. 3 (SEQ ID NOs:171-188) or FIG. 4 (SEQ IDNOs:189-192).
 95. An antibody or antibody fragment which recognizes andbinds to one or more peptides of amino acid sequences GNKYQTIDNYQPYPC(SEQ ID NO:118), LDGYSRRTTLSSKMYHTKGQEG (SEQ ID NO:119),RIQKDHHQASNSSRLHTCQRH (SEQ ID NO:120), RGEIEETITESFGND (SEQ ID NO:121),EIFQRCYCGEGLSCRIQKD (SEQ ID NO:122), MYWTDWVETPRIE (SEQ ID NO:123),MYWTDWGETPRIE (SEQ ID NO:124), KRTGGKRKEILSA (SEQ ID NO:125),ERVEKTTGDKRTRIQGR (SEQ ID NO:126), KQQCDSFPDCIDGSDE (SEQ ID NO:127), ora Dkk-1 amino acid sequence selected from the group consistingAsn34-His266 (SEQ ID NO:110), Asn34-Cys245 (SEQ ID NO:111), Asn34-Lys182(SEQ ID NO:112), Cys97-His266 (SEQ ID NO:113), Val139-His266 (SEQ IDNO:114), Gly183-His266 (SEQ ID NO:115), Cys97-Cys245 (SEQ ID NO:116), orVal139-Cys245 (SEQ ID NO:117).
 96. The antibody or antibody fragment ofclaim 95, wherein the antibody is a monoclonal antibody.
 97. Theantibody or antibody fragment of claim 95, wherein the antibody is apolyclonal antibody
 98. A method of identifying Dkk interacting proteinswhich modulate the interaction of Dkk with the Wnt signaling pathwaycomprising: (a) injecting Dkk and potential Dkk interacting protein mRNAinto a Xenopus blastomere; and (b) assessing axis duplication oranalyzing marker gene expression; and (c) identifying compositions whichelicit changes in axis duplication or marker gene expression as Dkkinteracting proteins which modulate the interaction of Dkk with the Wntsignaling pathway.
 99. The method of claim 98, wherein the mRNA of HBM,LRP5/6, any Wnt, Wnt antagonist, Wnt pathway modulator, or combinationof these is co-injected into the Xenopus blastomere.
 100. The method ofclaim 98, wherein the marker gene analyzed is Siamois, Xnr3, slug, Xbra,HNK-1, endodermin, Xlhbox8, BMP2, BMP4, XLRP6, EF-1, or ODC.
 101. Amethod for identifying Dkk interacting proteins which modulate theinteraction of Dkk with the Wnt signaling pathway comprising: (a)transfecting cells with constructs containing Dkk and potential Dkkinteracting proteins; and (b) assessing changes in expression of areporter gene linked to a Wnt-responsive promoter; and (c) identifyingas a Dkk interacting protein any protein which alters reporter geneexpression compared with cells transfected with a Dkk construct alone.102. The method of claim 101, wherein the cells are HOB-03-CE6, HEK293,or U2OS cells.
 103. The method of claim 101, wherein the Wnt-responsivepromoter is TCF or LEF.
 104. The method of claim 101, wherein the cellsare co-transfected with CMV -galactosidase.
 105. A method foridentifying compounds which modulate Dkk and LRP5/LRP6/HBM interactionscomprising: (a) immobilizing LRP5/LRP6/HBM to a solid surface; and (b)treating the solid surface with a secreted Dkk protein or a secretedepitope-tagged Dkk and a test compound; and (c) determining whether thecompound regulates binding between Dkk and LRP5/LRP6/HBM usingantibodies to Dkk or the epitope tag or by directly measuring activityof an epitope tag.
 106. The method of claim 105, wherein the epitope tagis alkaline phosphatase, histidine, or a V5 tag.
 107. A method foridentifying compounds which modulate the interaction of Dkk with the Wntsignaling pathway comprising: (a) transfecting cells with constructscontaining Dkk and Wnt proteins; (b) assessing changes in expression ofa reporter element linked to a Wnt- responsive promoter; and (c)identifying as a Dkk/Wnt interaction modulating compound any compoundwhich alters reporter gene expression compared with cells transfectedwith a Dkk construct alone.
 108. The method according to claim 107,wherein Wnt3a and Wnt1 constructs are co-transfected into the cells.109. The method according to claim 107, wherein the cells are U2-OS,HOB-03-CE6, or HEK293 cells.
 110. The method according to claim 107,wherein the reporter element used is TCF-luciferase, tk-Renilla, or acombination thereof.
 111. A method of testing compounds that modulateDkk-mediated activity in a mammal comprising (a) providing a group oftransgenic animals having (1) a regulatable one or more Dkk genes, (2) aknock-out of Dkk genes, or (3) a knock-in of one or more Dkk genes; (b)providing a second group of control animals respectively for the groupof transgenic animals in step (a); and (c) exposing the transgenicanimal group and control animal group to a potential Dkk-modulatingcompound which modulates bone mass or lipid levels; and (d) comparingthe transgenic animals and the control group of animals and determiningthe effect of the compound on bone mass or lipid levels in thetransgenic animals as compared to the control animals.
 112. A method ofscreening for compounds or compositions which modulate the interactionof Dkk and a Dkk interacting protein comprising: (a) exposing a Dkkinteracting proteins or a Dkk-binding fragment thereof to a compound;and (b) determining whether said compound binds to a Dkk interactingproteins or the Dkk-binding fragment thereof.
 113. The method of claim112, wherein said modulation is determined by whether said compoundbinds to the Dkk interacting protein or the Dkk-binding fragmentthereof.
 114. An antibody or antibody fragment which recognizes andbinds to a sequence depicted in FIG. 3 (SEQ ID NOs:171-188) or FIG. 4(SEQ ID NOs: 189-192).